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

Abstract

Irreversible fibrosis is a hallmark of myocardial infarction (MI) and heart failure. Extracellular matrix protein-1 (ECM-1) is up-regulated in these hearts, localized to fibrotic, inflammatory, and perivascular areas. ECM-1 originates predominantly from fibroblasts, macrophages, and pericytes/vascular cells in uninjured human and mouse hearts, and from M1 and M2 macrophages and myofibroblasts after MI. ECM-1 stimulates fibroblast-to-myofibroblast transition, up-regulates key fibrotic and inflammatory pathways, and inhibits cardiac fibroblast migration. ECM-1 binds HuCFb cell surface receptor LRP1, and LRP1 inhibition blocks ECM-1 from stimulating fibroblast-to-myofibroblast transition, confirming a novel ECM-1-LRP1 fibrotic signaling axis. ECM-1 may represent a novel mechanism facilitating inflammation-fibrosis crosstalk.

Keywords: extracellular matrix; fibroblasts; fibrosis; heart; inflammation; myocardial infarction.

Graphical abstract

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 al 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.

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.

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 set; 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) between ECM-1+ and ECM-1− cells (per cell subpopulation) within the major ECM-1-expressing cell subpopulations were assessed via MAST. Only M1MΦ, F-Act, F-SH, and F-SL returned lists of significant DEGs, and the top 5 up-regulated genes in ECM-1+ cells (x-axis) are shown in column graphs as fold change in gene expression (y-axis). (E) All up-regulated DEGs for M1MΦ, F-Act, F-SH, and F-SL were subject to Venn diagram analysis. No genes were commonly up-regulated in all 4 cell types; Fbln1 and Ccdc2 were commonly up-regulated in ECM-1+ F-Act, F-SH, and F-SL cells. Abbreviations as in Figures 1 and 2.

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 (IL)-1β2, IL-6, CCL2, transforming growth factor (TGF)-β1, TGF-β2, and collagen type I alpha 2 chain (Col1a2). Each gene was assessed via repeated-measures 2-way ANOVA (IL-6 and Col1a2 were assessed via repeated-measures mixed-effects analysis to account for missing values) with post hoc Šídák’s multiple comparisons test; expression of all genes are presented as the delta-delta threshold cycle (ΔΔCt) relative to Tpt1 housekeeping gene expression, n = 6/group. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. CT = control; other abbreviations as in Figures 1 and 2.

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 ANAT (ECM-1 as the response source) to visualize the ECM-1-dependent signaling network. The ANAT network for ECM-1 signaling in the “greater” PHOTON analysis direction (signaling score ≥6) is shown with ECM-1 highlighted in blue. All proteins shown in the network represent involvement in active mediation of ECM-1-dependent signal transduction. Pink pie charts represent fold changes of measured phosphorylation sites (see scale bar), with multiple phosphorylation sites represented by a pie chart divided by the number of phosphorylation sites measured. (C) ECM-1 binding partners were pulled down from a purified HuCFb membrane protein lysate, with the use of recombinant human ECM-1 protein as bait; 58 potential ECM-1-binding proteins were identified and 7 proteins of particular interest are presented: PTPN1, LAMB1, CTNND1, LRP1, FN1, LTBP2, and VCAN. (D) Immunofluorescence of HuCFb cells in culture showed that ECM-1 colocalizes with LRP1 (×40 magnification zoomed in); arrowhead shows an example area of interest (red = low-density lipoprotein receptor–related protein 1 [LRP1]; green = ECM-1; blue = DAPI/nuclei). (E) Immunofluorescence shows ECM-1 stimulates fibroblast-to-myofibroblast transition in HuCFbs at 48 hours of treatment (P = 0.003), and RAP (LRPAP1)–dependent LRP1 inhibition blocks this effect (ECM-1 vs ECM-1+RAP: P = 0.038; CT vs ECM-1+RAP; nonsignificant), presented as mean ± SD, analyzed by 1-way ANOVA with post hoc Šídák’s multiple comparisons test (n ≥ 9; ×2.5 magnification; green = α-SMA; red = vimentin; blue = DAPI/nuclei); #P < 0.05 comparing ECM-1+RAP and ECM-1 treatment groups. ∗P < 0.05; ∗∗P < 0.01. Abbreviations as in Figure 1, Figure 2, Figure 3, Figure 4.