Study On Action Mechanism Of TGF-β1/Smad Signaling In Thoracic Aortic Dissection

Objective:Thoracic aortic dissection (TAD) is life-threatened disease which is characterized with acute onset and high mortality. TAD is diagnosed in a growing number of people due to the advanced imaging with the incidence of 3-6/100000. About 50%-68% TAD patients die with conservative therapy, and the mortality rises up followed course of disease. The main treatments to TAD are surgery and intervention that are identified as longer time, more complication and restricted indication. Hence, it is considerable for us to explore new methods understanding prevention and treatment of TAD from pathogenesis of TAD.Recent studies have shown that the dysfunction of vascular smooth muscle cell (VSMC) means switching of VSMC play a role in the occurrence of TAD. There are two phenotype in VSMC, one is contractile phenotype with strong ability of maintain of mechanics of aortic, weak capability of proliferation and migration, another is synthetic phenotype with great capability of proliferation and migration, weak ability of maintain of mechanics of aortic. In general, VSMC exit with contractile phenotype but transmit into synthetic in TAD as relevant studies indicated.TGF-β1/Smad signaling play amount of function involved in differentiation, proliferation and apoptosis and it involve in the phenotype switching of VSMC. TGF-β1/Smad signaling acts via binding to some transcription factors. Several studies suggested that direct binding of TGF-β1-responsive Smads to the NANOG promoter plays an essential role in sustaining human embryonic stem cell (ESC) self-renewal, but this phenomenon has not been shown in TAD. So the aim of our research is to explore whether TGF-β1/Smad regulate the switching of VSMC via binding to Nanog promoter in TAD.Methods:The present study is divided into three sections.1. Collect the tissue of aorta from TAD patients (13 cases) and the autopsy people (10 cases) without disease of aorta. The expression of mRNA of TGF-β1, TGFβ-R1, Smad2, Smad3, Smad4 and Nanog are detected by RT-PCR. The expression of protein of TGF-β1, TGFβ-R1, Smad2, Smad3, Smad4 and Nanog are examined by Western Blot. The location and quantity of TGF-β1, TGFβ-R1, Smad2, Smad3, Smad4 and Nanog are detected by Immunohistochemical staining.2. Stimulating VSMC with TGF-β1 cytokine, then examine the difference of Smad2, Smad3 and a-SMA mRNA or protein expression by RT-PCR and Western Blot.3. Stimulating VSMC with TGF-β1 cytokine, then examine the difference of Nanog and a-SMA mRNA expression by RT-PCR and Western Blot, silencing the Nanog expression by SiRNA, then test the change of a-SMA mRNA expression by RT-PCR and Western Blot. At last, exploring whether Smad2 binding to the Nanog promoter by chromatin immunoprecipitation (CHIP).Result:1. The expression of TGF-β1, TGFβ-R1, Smad2, Smad3, Smad4 and Nanog. Both RT-PCR and Western Blot indicates that the expression of TGF-β1, TGFβ-R1, Smad2, Smad3, Smad4 and Nanog in TAD are higher than in control (P<0.05). Immunohistochemical staining shows that the expressions of TGF-β1, TGFβ-R1, Smad2, Smad3, Smad4 and Nanog in aorta are obvious.2. The relationship between TGF-β1/Smad and phenotype switching of VSMC. After stimulating of VSMC with TGF-β1 cytokine, the expressions of Smad2 and Smad3 mRNA are rising compared with non-TGF-β1 cytokine stimulating (P<0.05), the expression of a-SMA mRNA is decreasing compared with non-TGF-β1 cytokine stimulating (P<0.05).3. The study of phenotype switching of VSMC via Smad2 binding to Nanog promoter. After stimulating of VSMC with TGF-β1 cytokine, the expressions of Nanog mRNA rises compared with non-TGF-β1 cytokine stimulating (P<0.05), the expression of a-SMA mRNA decreases compared with non-TGF-β1 cytokine stimulating (P<0.05), then silencing the Nanog expression, the expression of a-SMA mRNA rises again. At last, the CHIP proved that Smad2 binding to the Nanog promoter from-298bp to -80bp.Conclusion:1. The expressions of TGF-β1、Smad、Nanog and relative factors in TAD are higher than in control indicate these factors are relative with TAD.2. TGF-β1 cytokine can stimulate the expression of Smad2 and Smad3, and the suppression of a-SMA suggest that TGF-β1, Smad2 and Smad3 lead to the phenotype switching of VSMC.3. It is confirmed that TGF-β1/Smad lead to the phenotype switching of VSMC by regulating the expression of Nanog via binding to the Nanog promoter in TAD.