Shi X, Guo L-W, Seedial S M, Si Y, Wang B, Takayama T, Suwanabol P A, Ghosh S, DiRenzo D, Liu B, Kent K C
Department of Surgery, University of Wisconsin, 1111 Highland Avenue, WIMR Building, Madison, WI 53705, USA.
Cell Death Dis. 2014 Jul 10;5(7):e1317. doi: 10.1038/cddis.2014.282.
We have previously shown that in the presence of elevated Smad3, transforming growth factor-β (TGF-β) transforms from an inhibitor to a stimulant of vascular smooth muscle cell (SMC) proliferation and intimal hyperplasia (IH). Here we identify a novel mechanism through which TGF-β/Smad3 also exacerbates IH by inhibiting SMC apoptosis. We found that TGF-β treatment led to inhibition of apoptosis in rat SMCs following viral expression of Smad3. Conditioned media from these cells when applied to naive SMCs recapitulated this effect, suggesting an autocrine pathway through a secreted factor. Gene array of TGF-β/Smad3-treated cells revealed enhanced expression of vascular endothelial growth factor (VEGF), a known inhibitor of endothelial cell apoptosis. We then evaluated whether VEGF is the secreted mediator responsible for TGF-β/Smad3 inhibition of SMC apoptosis. In TGF-β/Smad3-treated cells, VEGF mRNA and protein as well as VEGF secretion were increased. Moreover, recombinant VEGF-A inhibited SMC apoptosis and a VEGF-A-neutralizing antibody reversed the inhibitory effect of conditioned media on SMC apoptosis. Stimulation of SMCs with TGF-β led to the formation of a complex of Smad3 and hypoxia-inducible factor-1α (HIF-1α) that in turn activated the VEGF-A promoter and transcription. In rat carotid arteries following arterial injury, Smad3 and VEGF-A expression were upregulated. Moreover, Smad3 gene transfer further enhanced VEGF expression as well as inhibited SMC apoptosis. Finally, blocking either the VEGF receptor or Smad3 signaling in injured carotid arteries abrogated the inhibitory effect of Smad3 on vascular SMC apoptosis. Taken together, our study reveals that following angioplasty, elevation of both TGF-β and Smad3 leads to SMC secretion of VEGF-A that functions as an autocrine inhibitor of SMC apoptosis. This novel pathway provides further insights into the role of TGF-β in the development of IH.
我们之前已经表明，在Smad3升高的情况下，转化生长因子-β（TGF-β）从血管平滑肌细胞（SMC）增殖和内膜增生（IH）的抑制剂转变为刺激剂。在这里，我们确定了一种新机制，通过该机制TGF-β/Smad3还通过抑制SMC凋亡来加剧IH。我们发现，在病毒表达Smad3后，TGF-β处理导致大鼠SMC凋亡受到抑制。将这些细胞的条件培养基应用于未处理的SMC时，重现了这种效应，提示通过分泌因子的自分泌途径。TGF-β/Smad3处理细胞的基因芯片显示血管内皮生长因子（VEGF）表达增强，VEGF是一种已知的内皮细胞凋亡抑制剂。然后我们评估VEGF是否是负责TGF-β/Smad3抑制SMC凋亡的分泌介质。在TGF-β/Smad3处理的细胞中，VEGF mRNA和蛋白以及VEGF分泌均增加。此外，重组VEGF-A抑制SMC凋亡，而VEGF-A中和抗体逆转了条件培养基对SMC凋亡的抑制作用。用TGF-β刺激SMC导致形成Smad3和缺氧诱导因子-1α（HIF-1α）复合物，进而激活VEGF-A启动子和转录。在大鼠颈动脉损伤后，Smad3和VEGF-A表达上调。此外，Smad3基因转移进一步增强VEGF表达并抑制SMC凋亡。最后，在损伤的颈动脉中阻断VEGF受体或Smad3信号传导消除了Smad3对血管SMC凋亡的抑制作用。综上所述，我们的研究表明，血管成形术后，TGF-β和Smad3的升高导致SMC分泌VEGF-A，其作为SMC凋亡的自分泌抑制剂发挥作用。这一新途径为TGF-β在IH发展中的作用提供了进一步的见解。
