Objective Cardiac fibrosis is the accumulation of excess extracellular matrix in the heart, leading to worsened cardiac function and arrhythmia. Mice with macrophage-specific overexpression of urokinase plasminogen activator (SR-uPA+/0) have early cardiac macrophage accumulation in the heart and develop subsequent cardiac fibrosis. Macrophages activate or suppress fibroblast collagen production in response to stimuli such as growth factors, cytokines, and hypoxia. We hypothesized that excess uPA production by macrophages stimulates collagen production in fibroblasts, leading to excess collagen deposition in the heart.
Methods Murine embryonic fibroblast-derived NIH-3T3 cells were treated with SR-uPA+/0 and SR-uPA0/0 macrophage-conditioned media and were also cocultured with SR-uPA+/0 and SR-uPA0/0 macrophages. Some cells were also treated with transforming growth factor β1 (TGF-β1) as a positive control. RNA was extracted, and Northern blot for collagen I alpha1 (Col1) was performed. Cytokine assays were performed on SR-uPA+/0 and SR-uPA0/0 isolated peritoneal macrophage-conditioned media (± LPS) or heart explant culture using a Luminex-based assay of 20 major cytokines.
Results SR-uPA+/0 macrophage-conditioned media-treated NIH-3T3 cells had less Col1 mRNA than SR-uPA0/0-conditioned media-treated cells. NIH-3T3 cells cocultured with SR-uPA+/0 macrophages also showed less Col1 mRNA than those cocultured with SR-uPA0/0 macrophages. There was no significant difference in cytokine levels between SR-uPA+/0 and SR-uPA0/0 macrophages. In heart explant culture, SR-uPA+/0 mice had elevated levels of IL-6 compared with SR-uPA0/0 mice (13.6 [7.1-24.8] vs 4.5 [3-7] pg/mg tissue/18 h, p = .03), but none of the other 19 cytokines were significantly increased.
Conclusions Exposure of NIH-3T3 cells to SR-uPA+/0 macrophages or macrophage-conditioned media decreases transcription of collagen 1α1. IL-6 is increased in SR-uPA+/0 heart explant culture but not in isolated peritoneal macrophages. These data suggest that SR-uPA+/0 macrophages may secrete factors only in the heart that lead to increased fibroblast-mediated collagen production in the heart. Further studies are required to determine whether IL-6 specifically increases collagen production in cardiac fibroblasts and to elucidate its role in our model.