Department of Vascular Surgery, Shanghai Ninth People's Hospital Affiliated to Shanghai JiaoTong University School of Medicine, Shanghai, China; Vascular Center of Shanghai JiaoTong University, Shanghai, China.
J Surg Res. 2013 Oct;184(2):1182-95. doi: 10.1016/j.jss.2013.03.100. Epub 2013 Apr 18.
Deep venous thrombosis (DVT) confers vein wall injury associated with fibrosis and extracellular matrix turnover. The activation of vascular smooth muscle cells (VSMCs) and phenotypic switching are postulated to be the significant contributing factors in the evolution of the pathogenic processes. This study investigated the effect of tissue plasminogen activator (tPA) on the phenotypic switching and collagen deposition of VSMCs, as well as related signaling pathway that leads to this activation.
The model of stasis-induced DVT was established by ligation of the femoral vein. VSMCs transfected with the plasmid vector carrying a rat recombinant tPA gene with an enhanced green fluorescent protein (EGFP) tag (AdtPA-EGFP). Fibrotic change, expression of collagen type I, the cell number of media, and intimal thickness score were evaluated; the comparisons were made among the AdtPA-EGFP-transfected group, an empty vector (AdNull-EGFP) transfected group, and a phosphate-buffered saline perfused group in vivo. tPA induced VSMCs phenotypic switching and collagen deposition in vitro. The related signaling pathway molecules and the cell cycle progression were also investigated by western blot and flow cytometry.
In the AdtPA-EGFP stasis DVT model, early vein wall collagenolysis and deposition occurred more remarkable. Histological studies showed that the expression of vein wall collagen type I protein, cell number of media, and intimal thickness score was significantly increased (P < 0.05). In primary culture VSMCs, sustained stimulation with tPA induced collagen type I upregulation and triggered sequential signaling events involving Akt, extracellular signal-regulated kinases 1/2 (ERK1/2), glycogen synthase kinase-3 (GSK3)-β phosphorylation, and cyclin D1 induction. Blockade of phosphatidylinositol 3-kinase-Akt and ERK1/2 activation suppressed tPA-induced GSK3β phosphorylation, cyclin D1 expression, and the deposition of collagen type I.
tPA was a profibrotic factor that potentiated the phenotypic switching and the deposition of collagen in VSMC. The effect of tPA on VSMCs involved activation of Akt and ERK1/2 pathways and inhibition of GSK3β activity, which could promote a switch of the synthetic phenotype in VSMCs and lead to the remodeling of vascular injury.
深静脉血栓形成（DVT）会导致与纤维化和细胞外基质转换相关的静脉壁损伤。血管平滑肌细胞（VSMCs）的激活和表型转换被认为是导致致病过程的重要因素。本研究旨在探讨组织型纤溶酶原激活物（tPA）对 VSMCs 表型转换和胶原沉积的影响，以及导致这种激活的相关信号通路。
通过结扎股静脉建立淤滞性 DVT 模型。将携带大鼠重组 tPA 基因增强型绿色荧光蛋白（EGFP）标签的质粒载体转染 VSMCs（AdtPA-EGFP）。评估纤维性变化、胶原 I 型表达、中膜细胞数量和内膜厚度评分；并在 AdtPA-EGFP 转染组、空载体（AdNull-EGFP）转染组和磷酸盐缓冲液灌注组之间进行比较。tPA 在体外诱导 VSMCs 表型转换和胶原沉积。还通过 Western blot 和流式细胞术研究了相关信号通路分子和细胞周期进程。
在 AdtPA-EGFP 淤滞性 DVT 模型中，早期静脉壁胶原溶解和沉积更为明显。组织学研究表明，静脉壁胶原 I 型蛋白表达、中膜细胞数量和内膜厚度评分显著增加（P < 0.05）。在原代培养的 VSMCs 中，持续的 tPA 刺激诱导胶原 I 上调，并引发涉及 Akt、细胞外信号调节激酶 1/2（ERK1/2）、糖原合酶激酶 3-β（GSK3β）磷酸化和细胞周期蛋白 D1 诱导的连续信号事件。PI3K-Akt 和 ERK1/2 激活的阻断抑制了 tPA 诱导的 GSK3β 磷酸化、细胞周期蛋白 D1 表达和胶原 I 的沉积。
tPA 是一种促纤维化因子，可增强 VSMC 表型转换和胶原沉积。tPA 对 VSMCs 的作用涉及 Akt 和 ERK1/2 通路的激活和 GSK3β 活性的抑制，这可能促进 VSMCs 合成表型的转换，并导致血管损伤的重塑。
