Abstract 3345: Smad3 −/− Fibroblasts Are Hyperproliferative Cells With Impaired Migratory Capacity, Reduced Contractile Activity and Diminished Synthetic Function: Implications for Cardiac Repair and Remodeling
We have demonstrated that disruption of the TGF-β/Smad3 pathway attenuates post-infarction remodeling reducing cardiac fibrosis. Accordingly, we hypothesized that Smad3 signaling may critically regulate fibroblast function and gene expression in myocardial infarction. Reduced collagen deposition in Smad3 −/− infarcts was associated with a surprising increase in myofibroblast density. In comparison to WT animals, Smad3 −/− infarcts had a higher density of proliferating myofibroblasts. In vitro, TGF-β inhibited murine cardiac fibroblast proliferation; the antiproliferative effects of TGF-β were abrogated in Smad3 −/− fibroblasts. On the other hand, Smad3 signaling played an important role in fibroblast function. Smad3 −/− fibroblasts induced decreased collagen lattice contraction than WT cells; reduced contractile activity was associated with attenuated expression of α-smooth muscle actin. In addition, Smad3 −/− fibroblasts had decreased migratory activity upon stimulation with serum or TGF-β1 in a transwell migration assay. Smad3 absence significantly attenuated TGF-β1-induced synthesis of fibronectin, type I and III collagen. Because CTGF is an essential downstream mediator in TGF-β-induced fibrosis, we examined the role of the Smad3 pathway in TGF-β-mediated CTGF upregulation. CTGF was induced in healing infarcts after 24h-7days of reperfusion; late but not early CTGF upregulation was abrogated in Smad3 −/− infarcts. In addition, CTGF induction in TGF-β1-stimulated fibroblasts was dependent on Smad3. CTGF had modest effects on extracellular matrix protein expression by fibroblasts, but markedly enhanced TGF-β1-mediated actions suggesting a synergistic relationship between CTGF and TGF-β. The direct effects of CTGF and the synergistic actions between CTGF and TGF-β on cardiac fibroblasts were not dependent on Smad3. In the absence of Smad3, TGF-β-stimulated cardiac fibroblasts are hyperproliferative, but show attenuated migratory capacity and reduced matrix synthesis, are less effective in inducing wound contraction and exhibit decreased CTGF upregulation. CTGF appears to enhance the profibrotic effects of TGF-β in a Smad3-independent manner.