Tissue factor cytoplasmic domain exacerbates post-infarct left ventricular remodeling via orchestrating cardiac inflammation and angiogenesis

Abstract

The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and is implicated in cardiac pathology, the contribution of its cytoplasmic domain to post-infarct myocardial injury and adverse left ventricular (LV) remodeling remains unknown. Methods: Myocardial infarction was induced in wild-type mice or mice lacking the TF cytoplasmic domain (TF∆CT) by occlusion of the left anterior descending coronary artery. Heart function was monitored with echocardiography. Heart tissue was collected at different time-points for histological, molecular and flow cytometry analysis. Results: Compared with wild-type mice, TF∆CT had a higher survival rate during a 28-day follow-up after myocardial infarction. Among surviving mice, TF∆CT mice had better cardiac function and less LV remodeling than wild-type mice. The overall improvement of post-infarct cardiac performance in TF∆CT mice, as revealed by speckle-tracking strain analysis, was attributed to reduced myocardial deformation in the peri-infarct region. Histological analysis demonstrated that TF∆CT hearts had in the infarct area greater proliferation of myofibroblasts and better scar formation. Compared with wild-type hearts, infarcted TF∆CT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1) - Rac1 axis. In particular, infarcted TF∆CT hearts displayed markedly lower ratios of inflammatory M1 macrophages and reparative M2 macrophages (M1/M2). In vitro experiment with primary macrophages demonstrated that deletion of the TF cytoplasmic domain inhibited macrophage polarization toward the M1 phenotype. Furthermore, infarcted TF∆CT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Finally, the overall cardioprotective effects observed in TF∆CT mice could be abolished by subcutaneously infusing a cocktail of PAR1-activating peptide and PAR2-inhibiting peptide via osmotic minipumps. Conclusions: Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation.

Keywords: adverse left ventricular remodeling; angiogenesis; inflammation; myocardial infarction; tissue factor cytoplasmic domain.

Figure 1

Lack of the TF cytoplasmic domain promotes survival and protects cardiac function after MI. (A) The Kaplan-Meier survival curves. Log-rank test; n = 27 for WT mice and n = 21 for TF∆CT mice subjected to MI, n = 10 for WT and n = 9 for TF∆CT sham groups. (B and C) LVEF and LVIDs determined by echocardiography. *P < 0.05, ***p < 0.001 compared with WT mice by two-way ANOVA with Bonferroni post hoc test; n = 19 for WT mice and n = 19 for TF∆CT mice surviving MI, n = 10 for WT and n = 9 for TF∆CT sham groups. (D) Representative echocardiograms (M-mode tracing) illustrating cardiac changes after MI in WT versus TF∆CT mice. (E and F) Post-MI infarct size was estimated by measuring the length of myocardial infarct (in RED) and total length of LV endocardium (in CYAN) at the middle plane of the long-axis LV echocardiogram as indicated. Infarct size (%) = (length of infarct / length of LV endocardium) x 100. *P < 0.05, compared with WT, two-way ANOVA with Bonferroni post hoc test for multiple comparison. LVEF, left ventricular ejection fraction; LVIDs, internal diameter at end of systole.

Figure 2

Lack of the TF cytoplasmic domain preserves cardiac contractility and attenuates myocardium deformation after MI. (A) Three-dimensional (3D) regional wall velocity diagrams of LV endocardial strain illustrating contraction (red-orange) and relaxation (blue) of three consecutive cardiac cycles. (B) Vector diagrams illustrating the direction and magnitude of endocardial contraction at mid-systole. (C) A schematic diagram illustrating global and regional strain analysis. (D-G) Global and regional radial strain determined by speckle-tracking strain analysis. *P < 0.05, **p < 0.01 and ***p < 0.001 compared with WT mice by two-way ANOVA with Bonferroni post hoc test; n = 19 for WT mice and n = 19 for TF∆CT mice subjected to MI, n = 10 for WT and n = 9 for TF∆CT sham groups. Ant.Base, anterior base; Post.Base, posterior base, Ant.Mid, anterior middle; Post.Mid, posterior middle; Ant.Apex, anterior apex; Post.Apex, posterior apex.

Figure 3

Lack of the TF cytoplasmic domain enhances proliferation of myofibroblasts and endothelial cells after MI. (A) Representative heart setions (3 days post-MI) showing proliferation of Acellular matrix areas and expression of extracellular matrix proteins. Fibroblasts were stained by a-SMA and endothelial cells by IB4. Arrows indicate Ki67 positive fibroblasts and arrowheads indicate Ki67 positive endothelial cells. Images were taken by at 10x, 20x or 40x by Nikon Eclipse Ti-E inverted microscope. (B) Staining of a proliferative marker Ki67 at 3 days post-MI and myofibroblast marker α-SMA at 7 days post-MI. Scale bars represent 100 µM. (C and D) Quantification of Ki67 and α-SMA staining in the infarcted hearts. N = 5-6 per genotype per time-point. Mann-Whitney U test, *p < 0.05, **p < 0.01 compared with WT mice.

Figure 4

Lack of the TF cytoplasmic domain promotes post-MI cardiac extracellular matrix synthesis and scar formation. (A-D) Relative mRNA expression of Col1a1, Col3a1, TGFβ-1 and TIMP-1 was determined in the infarcted myocardium. Gene expression was normalized to sham-operated mice of its own genotype. N = 5-6 per genotype per time-point. (E) Representative heart sections (28 days post-MI) stained with Picrosirius Red, imaged under white light or polarized light. Scale bars represent 100 µM. (F) Quantification of collagen contents in the infarct 28 days post-MI (n = 14 per group). Mann-Whitney U test, *p < 0.05, **p < 0.01 compared with WT mice.

Figure 5

Lack of the TF cytoplasmic domain attenuates immune cell influx in response to MI. (A) Representative images of infarcted heart sections (3 days post-MI) stained for neutrophils, T cells and macrophages. Cell specific markers: Ly6G for neutrophils, CD3 for T-cells and MAC3 for macrophages. (B, C and D) Quantification of infiltrated cells in the infarct region. (E) Ratios of M1 to M2 macrophages in infarcted hearts (7 days post-MI). (F and G) Representative images of infarcted heart sections (7 days post-MI) stained for M1 (iNOS⁺) and M2 (CD206⁺) macrophages. N = 5 - 7 per genotype per time-point. Mann-Whitney U test, *p < 0.05, **p < 0.01, ***p < 0.001 compared with WT mice. Scale bars represent 100 µM.

Figure 6

Lack of the TF cytoplasmic domain decreases cytokine and chemokine production post-MI. Protein concentrations of cytokines and chemokines in lysates of infarcted myocardium were quantified by multiplex assay at different time-points post-MI. N = 5 - 6 per genotype per time-point; Kruskal-Wallis followed by Dunn post hoc test., *p < 0.05, **p < 0.01 at 3 days post-MI; ^ξp < 0.05, ^{ξ ξ}p < 0.01 at 7 days post-MI compared to WT mice.

Figure 7

Lack of the TF cytoplasmic domain promotes angiogenesis in infarcted myocardium. (A) Representative images of infarcted heart sections stained with DAPI (blue), WGA (green) and IB4 (red) at 7 days post-MI. (B and C) Quantification of blood vessel density and PAR2 staining in the infarct-and-border region. N = 5 - 7 per genotype per time-point; Mann-Whitney U test, *p < 0.05, **p < 0.01 compared with WT mice. (D) A representative heart section of a TF∆CT mouse at 28 days post-MI demonstrating co-localization of PAR2 with myocardial capillaries (stained with IB4). Panels (D', D'') show close-up of infarct border and infarct regions, respectively. An inset (D''') shows close-up of myocardial capillaries in the infarct region. White arrowheads indicate co-localization of PAR2 with capillaries. Scale bars: 100 µM.

Figure 8

The TF cytoplasmic domain regulates angiogenic signaling pathways in the infarcted heart. (A) A schematic diagram illustrating key angiogenic signaling molecules examined in the infarcted heart. (B-E) mRNA expression levels of PDGF-B and its receptor PDGFR-β, NOTCH ligand DLL4, SDF-1α receptor CXCR4, in the infarcted myocardium. Relative mRNA expression was normalized to sham-operated mice of its own genotype. N = 5 - 6 per genotype per time-point. (F) Western blot analysis of PDGF-B, PDGFR-β, DLL4 and CXCR4 protein levels in the infarcted myocardium from mice with SHAM surgery or 7 days post MI. (G) Quantification of protein levels analyzed by Western blot in panel F. N = 3 per genotype of mice. Protein levels were normalized to SHAM and presented as fold changes. Mann-Whitney U test, *p < 0.05, **p < 0.01 compared with WT mice; #p < 0.05 compared with sham. PDGF-B, platelet derived growth factor subunit B; PDGFR-β, PDGF-B receptor; DLL4, Delta Like Canonical Notch Ligand 4; SDF-1α, stromal cell-derived factor 1; CXCR4, C-X-C chemokine receptor type 4.

Figure 9

Synergistic activation of PAR1 and inhibition of PAR2 activity abolish cardioprotection in TFΔCT mice. (A) Experimental outline. TFΔCT mice, immediately prior to MI, were implanted with osmotic minipump supplemented with either the cocktail of PAR1 agonist and PAR2 antagonist peptides or the control peptide. Cardiac function was monitored by echocardiography. (B) The Kaplan-Meier survival curves. Log-rank test; n = 6 for TFΔCT mice infused with the control peptide, and n = 7 for TFΔCT mice infused with the cocktail of PAR1 agonist and PAR2 antagonist peptides, and n = 9 for WT mice serving as a control group. (C, D) LVEF and LVIDs determined by echocardiography for survived mice. **P < 0.01, ***p < 0.001 compared with TFΔCT mice infused with the control peptide by two-way ANOVA with Bonferroni post hoc test; n = 5 for both control peptide group and treatment group of TFΔCT mice, and n = 6 for WT mice. Of note, in panel D, the difference between WT mice and TFΔCT mice infused with the cocktail of PAR1 agonist and PAR2 antagonist peptides is not significant, but the difference between WT mice and TFΔCT mice infused with the control peptide was highly significant (p < 0.01).