Extracellular Matrix Protein-1 as a Mediator of Inflammation-Induced Fibrosis After Myocardial Infarction

Figures

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  Figure 1. ECM-1 Is Upregulated in Human Failing Hearts and Is Localized to Fibrotic Tissue, Leukocytes, and Endothelia (A) Western blotting shows that extracellular matrix protein (ECM)-1 protein is up-regulated in ischemic and dilated human heart failure patients, presented as mean ± SD, analyzed by means of Student’s t-test (n = 8/group). ∗P < 0.05. (B) Immunohistochemistry of ECM-1 protein expression in the human heart (NF: n = 4; heart failure: n = 8) shows that ECM-1 expression is predominantly interstitial, localized to fibrotic, inflammatory, and perivascular areas (×40 magnification; scale bars = 50 μm). (C) mRNA in situ hybridization (n = 3) confirms transcription of ECM-1 in these areas (×40 magnification zoomed in; scale bars = 50 μm). (D) ECM-1-specific analysis of single-cell/single-nuclear RNA sequencing dataset from Litviňuková et al22 identifies ECM-1 expression by fibroblasts, leukocytes, and vascular cells in the healthy human heart (gray = all cells; blue overlay = ECM-1+ cells). (E) The top 20 ECM-1-expressing cell subpopulations ranked by total number of ECM-1+ cells and by ECM-1 expression level (expressed as a Log2 average). NF = nonfailing.

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  Figure 2. Expression of ECM-1 in Cardiac Interstitial Cells Before and After MI (A) UMAP plot showing cell populations in single-cell RNA sequencing of cardiac interstitial cells from sham-operated and MI hearts combined, and (B) faceted according to condition. (C) ECM-1 expression on UMAP coordinates according to condition. (D, E, F) Violin plots of ECM-1 expression according to condition for cell subpopulations in (D) fibroblasts, (E) endothelial (EC) and mural cells, and (F) monocyte (Mo) and macrophage (MΦ) populations. (G) UMAP plot of Mo/MΦ cell populations at day 3 after MI. (H, I) ECM-1 expression in day-3 post-MI Mo/MΦ cells on (H) UMAP coordinates and (I) in violin plots.Abbreviations as in Figure 1.

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  Figure 3. ECM-1 Expression Strongly Correlated With the Number of Outbound Ligand-Receptor Interactions Between Cells, Extracellular Matrix Organization, and Cell Adhesion Genes (A) Per-cell Spearman correlation analysis of ECM-1 expression against all genes in the mouse single-cell RNA sequencing data set20; all P <0.001. The top 10 positively correlated genes (x-axis) are shown as column graphs of Spearman’s r-value (y-axis). The top ECM-1-correlated genes (with Spearman’s r > 0.30) were subject to gene ontology biological process (GOBP) enrichment analysis via the GOnet/DICE online tool. The top 10 GOBPs are presented as a bar chart ranked by false discovery rate (FDR) P value. (B) The average ECM-1 expression level (Log2) of each cell subpopulation is positively correlated with the number of significant outbound ligand-receptor connections (r = 0.719; P < 0.001), analyzed by Pearson’s correlation and simple linear regression. (C) ECM-1, Lgals1, and Lrp1 expression overlayed on the UMAP plot of cardiac interstitial cells from sham and MI (as shown in Figure 2A), to compare the cellular expression profile of the top 2 ECM-1 correlated genes. (D) Differentially expressed genes (DEGs)…

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  Figure 4. ECM-1 Inhibits Human Cardiac Fibroblast Cell Migration in Culture, and Stimulates Fibroblast-to-Myofibroblast Transition and Inflammatory and Fibrotic Signaling Pathways in HuCFbs (A) ECM-1 treatment (20 ng/mL) significantly reduced the migration rate of HuCFb cells over 24 hours relative to control subjects (n = 4/group), as assessed via both repeated-measures 2-way analysis of variance (ANOVA) with post hoc Šídák’s multiple comparisons test (P = 0.044) and inclination of the linear equation of growth rate via Student’s t-test (P = 0.027). ECM-1 had no effect on cell proliferation assessed via MTT assay after 24, 48, or 72 hours of treatment, as assessed via repeated-measures 2-way ANOVA with post hoc Šídák’s multiple comparisons test. (B) Immunofluorescence shows ECM-1 stimulates fibroblast-to-myofibroblast transition in HuCFbs after 48 hours of treatment, as analyzed by Student’s t-test (P = 0.014; n = 6; ×2.5 magnification; green = α-smooth muscle actin [SMA]; red = vimentin; blue = DAPI/nuclei). (C) HuCFbs were treated with ECM-1 (20 ng/mL) or medium alone for 3, 6, or 16 hours and assessed via quantitative polymerase chain reaction for mRNA expression of Wnt5a, interleukin…

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  Figure 5. Proteomics Reveals ECM-1-HuCFb Cell Membrane Binding Partners and Downstream Signaling Mechanisms: ECM-1 Binds LRP1 Cell Surface Receptor, Which Is Required for ECM-1-Dependent Stimulation Of Profibrotic Fibroblast-to-Myofibroblast Transition (A) HuCFb cells were treated with ECM-1 (20 ng/mL) for 10 minutes and subject to phosphoproteomics (Ser/Thr) mass spectrometry (titanium dioxide workflow; n = 6/group). Thirty-eight proteins with significantly different phosphorylation levels were identified between CT (medium alone) and ECM-1-treated cells, as assessed via Student’s t-test with permutation-based FDR; data are presented as the Log2 difference (CT − ECM-1) in phosphorylation level; green = down-regulated; red = up-regulated. Note that proteins measured multiple times represent different phosphosites within those proteins. Fisher’s exact enrichment testing was conducted on significantly up-regulated and down-regulated phosphorylation sites in ECM-1-treated samples (separately), and 4 of the most over-represented GOBPs are shown. (B) Our list of differential phosphorylation sites in ECM-1-treated HuCFbs was then subjected to PHOTON pathway analysis (2-sided) with reconstruction…