Impaired non-canonical transforming growth factor-ß signalling prevents profibrotic phenotypes in cultured peptidylarginine deiminase 4-deficient murine cardiac fibroblasts.

Transforming growth factor-ß (TGF-ß) becomes rapidly activated in the infarcted heart. Hence, TGF-ß-mediated persistent activation of cardiac fibroblasts (CFs) and exaggerated fibrotic responses may result in adverse cardiac remodelling and heart failure. Additionally, peptidylarginine deiminase 4 (PAD4) was described to be implicated in organ fibrosis. Here, we investigated the impact of PAD4 on CF function and myofibroblast transdifferentiation in vitro. The expression of fibrosis-related genes was largely similar in cultured WT and PAD4-/- CFs of passage 3, although collagen III was reduced in PAD4-/- CFs. Exposure to TGF-ß inhibited proliferation and increased contractile activity and migration of WT CFs, but not of PAD4-/- CFs. However, under baseline conditions, PAD4-/- CFs showed comparable functional characteristics as TGF-ß-stimulated WT CFs. Although the SMAD-dependent TGF-ß pathway was not disturbed in PAD4-/- CFs, TGF-ß failed to activate protein kinase B (Akt) and signal transducer and activator of transcription 3 (STAT3) in these cells. Similar results were obtained in WT CFs treated with the PAD4 inhibitor Cl-amidine. Abrogated Akt activation was associated with diminished levels of phosphorylated, inactive glycogen synthase kinase-3ß (GSK-3ß). Consequently, PAD4-/- CFs did not upregulate collagen I and α-smooth muscle actin (α-SMA) expression after TGF-ß treatment. Thus, PAD4 is substantially involved in the regulation of non-canonical TGF-ß signalling and may represent a therapeutic target for the treatment of adverse cardiac remodelling.

Compromised Anti-inflammatory Action of Neutrophil Extracellular Traps in PAD4-Deficient Mice Contributes to Aggravated Acute Inflammation After Myocardial Infarction.

Innate immune responses and rapid recruitment of leukocytes, which regulate inflammation and subsequent healing, play a key role in acute myocardial infarction (MI). Peptidylarginine deiminase 4 (PAD4) is critically involved in chromatin decondensation during the release of Neutrophil Extracellular Traps (NETs) by activated neutrophils. Alternatively, activated macrophages (M2) and accurate collagen deposition determine the repair of the infarcted heart. In this study, we investigated the impact of NETs on macrophage polarization and their role for acute cardiac inflammation and subsequent cardiac healing in a mouse model of acute MI. NETs were found to promote in vitro macrophage polarization toward a reparative phenotype. NETs suppressed pro-inflammatory macrophages (M1) under hypoxia and diminished IL-6 and TNF-α expression. Further on, NETs strongly supported M2b polarization and IL-10 expression. In cardiac fibroblasts, NETs increased TGF-ß expression under hypoxic culture conditions. PAD4-/- mice subjected to permanent ligation of the left anterior descending artery suffered from overwhelming inflammation in the acute phase of MI. Noteworthy, PAD4-/- neutrophils were unable to release NETs upon ex vivo stimulation with ionomycin or PMA, but produced significantly higher amounts of reactive oxygen species (ROS). Increased levels of circulating cell-free DNA, mitochondrial DNA and cardiac troponin were found in PAD4-/- mice in the acute phase of MI when compared to WT mice. Reduced cardiac expression of IL-6, IL-10, and M2 marker genes, as well as increased TNF-α expression, suggested a pro-inflammatory state. PAD4-/- mice displayed significantly increased cardiac MMP-2 expression under baseline conditions. At day 1, post-MI, PAD4-/- mice showed increased end-diastolic volume and increased thinning of the left ventricular wall. Interestingly, improved cardiac function, as demonstrated by significantly increased ejection fraction, was found at day 21. Altogether, our results indicate that NETs support macrophage polarization toward an M2 phenotype, thus displaying anti-inflammatory properties. PAD4 deficiency aggravates acute inflammation and increases tissue damage post-MI, partially due to the lack of NETs.