Abstract 227: TGF-β/Smad3 Promotes Smooth Muscle Cell De-differentiation and Proliferation Through Crosstalk with the Wnt/β-Catenin Pathway

Abstract

Introduction: Endovascular interventions are the first line treatment for patients with atherosclerotic disease. However, a significant number of these fail due to neointimal hyperplasia. The Wnt4/β-catenin pathway has been reported to play an important role in this process, but the mechanism underlying the up-regulation of this pathway after injury remains unclear. Our lab has previously demonstrated that following vascular injury, elevated TGF-β and its signaling protein, Smad3, exacerbate intimal hyperplasia in Smooth Muscle Cells (SMCs). Moreover, our Affymetrix array data revealed an upregulation (>3 fold) of several members of the Wnt family in response to TGF-β/Smad3 treatment of SMCs. Therefore, we hypothesize that the TGF-β/Smad3 axis is responsible for the activation of the Wnt/β-catenin signaling pathway that promotes neointimal hyperplasia. Methods and Results: To mimic the injury induced up-regulation of TGF-β/Smad3 in vitro, SMCs were infected with an adenovirus to increase Smad3 and then treated with recombinant TGF-β for 48 hrs. Real time PCR confirmed that Wnt expression increased substantially in response to TGF-β/Smad3 stimulation. Using western blotting, we found that SMCs treated with TGF-β/Smad3 had a significantly increased stabilization of β-catenin compared to SMCs treated with TGF-β alone. This effect was abolished by Niclosamide, a broad-spectrum Wnt inhibitor. Conversely, β-catenin stabilization increased after stimulation by a Wnt agonist and recombinant Wnts 2b, 4, 5a, and 9a, but not Wnt11. Importantly, we found that these Wnt ligands stimulated the expression of SMC de-differentiation markers including CXCR4, CD34, Sox18, and FGF1 to varying levels as measured by RT-PCR. Wnt4 and Wnt5a were also found to significantly promote SMC proliferation (21% and 27%, respectively, p<0.05) when measured by the Cell Titer Glo Assay. Conclusions: These results suggest that the neointima-producing Wnt/β-catenin pathway may be activated due to elevated TGF-β/Smad3 signaling and promote SMC de-differentiation and proliferation. Thus, the cross talk of these two pathways may provide a potential therapeutic target for preventing failure of endovascular interventions.