The Development of Myocardial Fibrosis in Transgenic Mice With Targeted Overexpression of Tumor Necrosis Factor Requires Mast Cell–Fibroblast Interactions

Abstract

Background Transgenic mice with cardiac restricted overexpression of tumor necrosis factor (MHCsTNF mice) develop progressive myocardial fibrosis, diastolic dysfunction and adverse cardiac remodeling. Insofar as tumor necrosis factor (TNF) does not directly stimulate fibroblast collagen synthesis, we asked whether TNF-induced fibrosis was mediated indirectly through interactions between mast cells and cardiac fibroblasts. Methods and Results Cardiac mast cell number increased 2–3-fold (p < 0.001) in MHCsTNF mice compared to littermate (LM) controls. Outcrossing MHCsTNF mice with mast cell deficient (c-kit−/−) mice showed that the 11-fold increase (p < 0.001) in collagen volume fraction in MHCsTNF/c-kit+/− mice was abrogated in MHCsTNF/c-kit−/− mice, and that the leftward shifted LV pressure-volume curve in the MHCsTNF/c-kit+/− mice was normalized in the MHCsTNF/c-kit−/− hearts. Furthermore, the increase in TGF-β1 and type I TGF-β receptor (TβR I) mRNA levels was significantly (p = 0.03, p = 0.01 respectively) attenuated in MHCsTNF/c-kit−/− when compared to MHCsTNF/c-kit+/− mice. Co-culture of fibroblasts with mast cells resulted in enhanced α-smooth muscle actin expression, increased proliferation and collagen mRNA expression, and increased contraction of 3-D collagen gels in MHCsTNF fibroblasts compared to LM fibroblasts. The effects of mast cells were abrogated by TβR I antagonist NP-40208. Conclusions These results suggest that increased mast cell density with resultant mast cell-cardiac fibroblast cross-talk is required for the development of myocardial fibrosis in inflammatory cardiomyopathy. Cardiac fibroblasts exposed to sustained inflammatory signaling exhibit an increased repertoire of pro-fibrotic phenotypic responses in response to mast cell mediators.

Necrotic Myocardial Cells Release Damage‐Associated Molecular Patterns That Provoke Fibroblast Activation In Vitro and Trigger Myocardial Inflammation and Fibrosis In Vivo

“…The proliferation of primary cardiac fibroblasts and NIH/3T3 fibroblast proliferation was determined by measuring bromodeoxyuridine (5-bromo-2′-deoxyuridine [BrdU]) incorporation as we have described previously,9 using a commercially available assay (Colorimetric Cell Proliferation ELISA, Roche). The fibroblast cell cultures were treated for 48 hours with PBS, NMC supernatants, or supernatants from necrotic liver cells (NLCs), heat-denatured NMCs and LMCs, NMCs and LMCs treated with DNase (0.5 μg/mL) or RNase (0.05 μg/mL), or 0.1 to 1000 ng/mL recombinant DAMPs (HMGB1 [Sigma-Aldrich], galectin-3 [R&D systems], IL-1α [Cell Signaling], S100β [Sigma-Aldrich], S100A8/A9 [ProSpec-Tany TechnoGene Ltd]) maintained in serum-free DMEM (Sigma-Aldrich), followed by the addition of BrdU for an additional 24 hours.…”

“…The α–smooth muscle actin (SMA) expression was determined by performing flow cytometric analysis of NIH/3T3 fibroblast cultures that had been stimulated for 48 hours with diluent or NMC supernatants (10 μg/mL), exactly as described earlier 9. The extent of α-SMA staining in the fibroblast cultures was expressed as the mean fluorescence intensity (MFI).…”

“…At 72 hours after surgical recovery, the mice were killed, the hearts were harvested, and paraffin sections were made. Slides were stained with hematoxylin and eosin and picrosirius red acid, as described previously 9. The degree of myocardial inflammation and myocardial fibrosis were quantified by computer-assisted densitometric analysis of the percentage LV apical myocardium with leukocytic infiltration (×50) or picrosirius red staining (×100).…”

Innate Immunity and the Failing Heart

“…29 These studies demonstrated that sustained myocardial inflammation leads to temporal changes in the balance between MMP activity tissue inhibitor of matrix metalloproteinases (TIMPs) and mast cell-mediated TGF-β signaling. 30 Collectively, these time-dependent changes favor degradation of the extracellular matrix during the onset of inflammation and progressive myocardial fibrosis following sustained inflammation. Thus, the sustained activation of inflammatory signaling contributes to LV remodeling through a variety of different mechanisms that involve both the myocyte and non-myocyte components of the myocardium.…”

Mechanical ventilation augments bleomycin-induced epithelial–mesenchymal transition through the Src pathway

“…5 The following primers were used for real-time polymerase chain reaction (PCR): collagen 1a1, forward primer 5 0 -CCAAGGGTAACAGCGGTGAA-3 0 and reverse primer 5 0 -CCTCGTTTTCCTTCTTCTCCG-3 0 ; collagen 1a2, forward primer 5 0 -TGTTGGCCCATCTGGTAAAGA-3 0 and reverse primer 5 0 -CAGGGAATCCGATGTTGCC-3 0 ; collagen 3a1, forward primer 5 0 -TCAAGTCTGGAGTGGGAGG-3 0 and reverse primer 5 0 -TCCAGGATGTCCAGAAGAACC-3 0 ; and GAPDH as internal control, forward primer 5 0 -AATGCATCC TGCACCACCAA-3 0 and reverse primer 5 0 -GTAGCCATATTC ATTGTCATA-3 0 (Integrated DNA Technologies, Coralville, IA, USA). 28 All quantity PCR reactions using SYBR Master Mix were performed on an ABI Prism 7000 sequence detector PCR system (Applied Biosystems, Foster City, CA, USA).…”