Targeting myeloid cell coagulation signaling blocks MAP kinase/TGF-β1–driven fibrotic remodeling in ischemic heart failure
Venkata Garlapati, … , Wolfram Ruf, Philip Wenzel
Venkata Garlapati, … , Wolfram Ruf, Philip Wenzel
Published December 22, 2022
Citation Information: J Clin Invest. 2023;133(4):e156436. https://doi.org/10.1172/JCI156436.
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Research Article Immunology

Targeting myeloid cell coagulation signaling blocks MAP kinase/TGF-β1–driven fibrotic remodeling in ischemic heart failure

Abstract

Despite major advances in acute interventions for myocardial infarction (MI), adverse cardiac remodeling and excess fibrosis after MI causing ischemic heart failure (IHF) remain a leading cause of death worldwide. Here we identify a profibrotic coagulation signaling pathway that can be targeted for improved cardiac function following MI with persistent ischemia. Quantitative phosphoproteomics of cardiac tissue revealed an upregulated mitogen-activated protein kinase (MAPK) pathway in human IHF. Intervention in this pathway with trametinib improves myocardial function and prevents fibrotic remodeling in a murine model of non-reperfused MI. MAPK activation in MI requires myeloid cell signaling of protease-activated receptor 2 linked to the cytoplasmic domain of the coagulation initiator tissue factor (TF). They act upstream of pro-oxidant NOX2 NADPH oxidase, ERK1/2 phosphorylation, and activation of profibrotic TGF-β1. Specific targeting with the TF inhibitor nematode anticoagulant protein c2 (NAPc2) starting 1 day after established experimental MI averts IHF. Increased TF cytoplasmic domain phosphorylation in circulating monocytes from patients with subacute MI identifies a potential thromboinflammatory biomarker reflective of increased risk for IHF and suitable for patient selection to receive targeted TF inhibition therapy.

Authors

Venkata Garlapati, Michael Molitor, Thomas Michna, Gregory S. Harms, Stefanie Finger, Rebecca Jung, Jeremy Lagrange, Panagiotis Efentakis, Johannes Wild, Maike Knorr, Susanne Karbach, Sabine Wild, Ksenija Vujacic-Mirski, Thomas Münzel, Andreas Daiber, Moritz Brandt, Tommaso Gori, Hendrik Milting, Stefan Tenzer, Wolfram Ruf, Philip Wenzel

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Figure 8

TF cytoplasmic tail deletion attenuates ROS production and ERK1/2–TGF-β1 signaling–dependent cardiac fibrosis and improves cardiac function.

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TF cytoplasmic tail deletion attenuates ROS production and ERK1/2–TGF-β1...
WT and TFΔCT mice were subjected to permanent LAD ligation versus sham surgery and investigated after 7 days and 4 weeks. (A) Confocal microscopy of myocardial cryosections. Representative images and quantification of MFI of CD45/gp91phox double-positive cells. Scale bars: 75 μm. (B) Assessment of superoxide formation in infarcted myocardium by dihydroethidium-HPLC analysis. Representative chromatogram of 2-hydroxyethidium (2-HE), the oxidation product of DHE, and quantification normalized to weight of the infarcted tissue. Ordinary 1-way ANOVA, Šidák’s multiple-comparison test; n = 5–6 animals per group. (C) Protein expression analysis of monocytes isolated from NOX2–/– animals and stimulated with an inflammatory cytokine cocktail containing IL-6, TNF-α, and CCL2 at a concentration of 20 ng/mL with and without hypoxia for 4 hours. Western blotting of p-ERK1/2 (normalized to ERK1/2) and activated TGF-β1 (normalized to GAPDH). Ordinary 1-way ANOVA, Šidák’s multiple-comparison test; n = 4 replicates per group (2–3 mice were pooled for each sample). (D) Western blot analysis of activated TGF-β1 (normalized to GAPDH) and p-SMAD2 (normalized to total SMAD2) in infarcted myocardium obtained from WT or TFΔCT mice after 7 days of MI. Representative blots and quantification of biological replicates. Ordinary 1-way ANOVA, Šidák’s multiple-comparison test; n = 5–7 animals per group. (E) Sirius red staining and deconvoluted images of fibrotic area on paraffin-embedded heart sections 4 weeks after permanent LAD ligation to induce IHF versus sham surgery. Representative images and quantification of fibrotic areas normalized to surface area. Ordinary 1-way ANOVA, Šidák’s multiple-comparison test; n = 5–7 animals per group. (F) Longitudinal echocardiographic studies over 4 weeks for LVEF (%) and LVEDV (μL) in parasternal long axis M-mode. Two-way ANOVA, Bonferroni’s multiple-comparisons test; n = 6–17 animals per group. (G) Kaplan-Meier survival analysis of permanently LAD-ligated versus sham-operated C57BL/6J and TFΔCT mice over 4 weeks. Log-rank (Mantel-Cox) test; n = 15–20 animals per group. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.