S. Franco1, X. Shi1, L. Guo1, D. Direnzo1, C. Kent1 1University Of Wisconsin,Surgery,Madison, WI, USA
Introduction: Restenosis is the re-narrowing of the vessel lumen post vascular reconstructive procedures such as balloon angioplasty and bypass and occurs in ~50% of patients undergoing these procedures. Intimal hyperplasia (IH) is a complex process involving smooth muscle cell (SMC) migration and proliferation, and is the primary contributor to restenosis. SOX18 is a transcription factor important in developmental processes. It was previously shown that down regulation of SOX18 prevents proliferation and reduces migration in in cultured SMCs that were stimulated by serum. However, the role of SOX18 in the development of IH is not known. What factors regulate SOX18 in this process and the mechanism by which SOX18 regulates pro-IH SMC behaviors are poorly defined. We have previously shown that elevated TGFβ and its signaling protein Smad3 in injured arteries stimulate SMC proliferation and migration and IH. These results led us to hypothesize that TGFβ/Smad3 regulate SOX18 expression, resulting in enhanced SMC proliferation and migration, and IH following injury.
Methods: To determine the expression of Sox18 and Smad3 in vivo, carotid artery balloon injury was performed in male Sprague-Dawley rats to induce IH/restenosis, and immunohistochemistry was then carried out using anti-SOX18 and anti-Smad3 antibodies on sections from injured carotid arteries or uninjured controls collected at 3, 7, or 14 days post injury. In order to mimic up-regulation of Smad3 and TGFβ in vivo, primary rat aortic SMCs were infected with adenovirus expressing Smad3 (or GFP control) and then stimulated with TGFβ (5ng/ml) or solvent for 24 hours. Total RNA and protein were extracted and used for microarray analysis, real-time PCR, and western blotting.
Results: Using immunohistochemistry we observed that Sox18 positive cells (versus total cells) were substantially increased in the neointima layer of balloon injured rat carotid arteries at 7 and 14 days post-angioplasty compared to those in the media layer and uninjured control. Smad3 expression was up-regulated in a similar pattern. Our Affymetrix gene expression array data revealed TGFβ/Smad3-stimulated differential expression of multiple genes including Sox18 which was up-regulated 3.19 fold (p=0.02). Real time PCR confirmed Sox18 up-regulation (28 fold, p<0.01) in response to TGFβ/Smad3 stimulation compared to control. Furthermore, Western blot results demonstrated Sox18 protein was also up-regulated following TGFβ/Smad3 stimulation.
Conclusion: In conclusion, we observed up-regulation of Sox18 in neointima following arterial injury where Smad3 is also increased. We confirmed that Sox18 up-regulation was stimulated by TGFβ/Smad3 in SMCs in vitro. Further studies are warranted to investigate a possible mechanism of Sox18-mediated IH stimulated by TGFβ/Smad3 up-regulation.