Abstract 11268: Damage Associated Molecular Patterns (DAMPs) Activate Cardiac Fibroblasts Through Toll-Like Receptor 4: Implications for Myocardial Fibrosis
Background: Necrotic cell death provokes an inflammatory response secondary to the release of damage associated molecular patterns (DAMPs) from dying cells, which then activate innate immune signaling pathways. Based on the known relationship between necrotic cell death and cardiac fibrosis we asked whether DAMPs from the cardiac myocytes (c-DAMPs) would provoke fibroblast proliferation and activation through innate immune signaling.
Methods and Results: c-DAMPs were prepared from myocardial cytosolic extracts obtained by repetitive freeze thaw cycles of finely minced hearts. Western blotting of the myocardial cytosolic extracts revealed several known DAMPs: including HMGB1, Galectin-3, S100A8 and S100A9. Treatment of NIH/3T3 and mouse primary cardiac fibroblasts with increasing concentrations of extracts containing c-DAMPs revealed a concentration dependent 1.25-1.35 fold increase (p < 0.01) in fibroblast proliferation. Similar increases in fibroblast proliferation were also observed following treatment with individual purified DAMPs (HMGB1, Galectin-3, S100A8 and S100A9). Notably, heat denatured c-DAMP extracts had no effect on fibroblast proliferation. Treatment of NIH/3T3 fibroblasts with c-DAMPs resulted in activation of Akt, ERK, and c-Jun signaling pathways, but had no effect on p38 activation. Co-treatment of c-DAMP stimulated fibroblasts with Akt and ERK inhibitors abrogated the c-DAMP induced increase in fibroblast proliferation. Remarkably, the effect of c-DAMPs on fibroblast proliferation was prevented completely by a neutralizing antibody directed against Toll-like receptor 4 (TLR4), an innate immune pattern recognition receptor present on fibroblasts. Importantly, in addition to promoting proliferation, c-DAMPs also provoked fibroblast activation, as denoted by increased (p < 0.05) alpha-smooth muscle actin expression and increased (p < 0.05) collagen1A1 and 3A1 gene expression.
Conclusion: Taken together, these results suggest that the release of cytosolic proteins (c-DAMPs) from necrotic cardiac myocytes provokes a TLR4-mediated increase in fibroblast proliferation and collagen synthesis, thus providing a critical link between necrotic cell death and the development of myocardial fibrosis.
- © 2012 by American Heart Association, Inc.