Section of Vascular Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden.
J Vasc Surg. 2010 Jul;52(1):167-75. doi: 10.1016/j.jvs.2010.02.282.
Vascular smooth muscle cells (SMCs) are exposed to fluid shear stress (FSS) after interventional procedures such as balloon-angioplasty. Whereas the effects of hemodynamic forces on endothelial cells are explored in detail, the influence of FSS on smooth muscle cell function is poorly characterized. Here, we investigated the effect of FSS on SMC gene expression and function.
Laminar FSS of arterial level (14 dynes/cm(2)) was applied to SMC cultures for 24 hours in a parallel-plate flow chamber. The effect of FSS on gene expression was first screened with microarray technology, and results further verified by real time polymerase chain reaction (RT-PCR) and immunoblotting. Tissue factor pathway inhibitor-2 (TFPI-2) and caspase-3 protein expression was studied in the rat carotid artery after balloon-injury, and the effect of TFPI-2 on SMC DNA synthesis and apoptosis was examined in vitro.
Microarrays identified TFPI-2 as one of the most differentially expressed gene by FSS in cultured SMCs (P < .001). Gene set enrichment analysis revealed significant regulation of genes linked to proliferation, apoptosis, and cell cycle regulation. TFPI-2 induction was confirmed by RT-PCR and immunoblotting demonstrating a more than 400-fold (P < .001) increase in TFPI-2 mRNA in SMCs exposed to FSS compared with static controls, and a consistent protein upregulation. Functionally, SMC proliferation was decreased by FSS (P < .001), and recombinant TFPI-2 was found to inhibit SMC proliferation (P < .001) and induce SMC apoptosis as indicated by activation of caspase-3 (P < .01). In vivo, TFPI-2 expression was found to be upregulated 5, 10, and 20 hours (P < .01) after rat carotid balloon injury, and immunohistochemistry demonstrated TFPI-2 protein in FSS-exposed luminal SMCs, co-localized with caspase-3 in the rat carotid neointima.
FSS influenced gene expression associated with cell growth and apoptosis in cultured SMCs and strongly induced expression of TFPI-2 mRNA and protein. TFPI-2 was expressed in luminal, FSS-exposed SMCs together with caspase-3 in the rat carotid neointima after balloon injury. Functionally, TFPI-2 may play a role in vessel wall repair by regulating SMC proliferation and survival. Further studies are needed to elucidate the mechanisms by which TFPI-2 controls SMC function.
血管平滑肌细胞（SMCs）在球囊血管成形术等介入手术后会受到流体切应力（FSS）的影响。虽然血流动力对内皮细胞的影响已被详细研究，但 FSS 对平滑肌细胞功能的影响还知之甚少。在这里，我们研究了 FSS 对 SMC 基因表达和功能的影响。
在平行板流动腔中，用动脉水平的层流 FSS（14 达因/平方厘米）对 SMC 培养物施加 24 小时。首先用微阵列技术筛选 FSS 对基因表达的影响，并用实时聚合酶链反应（RT-PCR）和免疫印迹进一步验证。研究了球囊损伤后大鼠颈动脉组织因子途径抑制剂-2（TFPI-2）和半胱天冬酶-3 蛋白的表达，并在体外研究了 TFPI-2 对 SMC DNA 合成和凋亡的影响。
微阵列将 TFPI-2 鉴定为 FSS 在培养的 SMC 中最差异表达的基因之一（P <.001）。基因集富集分析显示，与增殖、凋亡和细胞周期调节相关的基因受到显著调控。RT-PCR 和免疫印迹证实 TFPI-2 诱导，与静态对照相比，暴露于 FSS 的 SMC 中 TFPI-2 mRNA 增加了 400 多倍（P <.001），并一致上调了蛋白。功能上，FSS 降低了 SMC 的增殖（P <.001），并且发现重组 TFPI-2 抑制 SMC 增殖（P <.001）并诱导 SMC 凋亡，如半胱天冬酶-3 的激活所示（P <.01）。体内，TFPI-2 的表达在大鼠颈动脉球囊损伤后 5、10 和 20 小时（P <.01）升高，免疫组织化学显示 TFPI-2 蛋白在 FSS 暴露的管腔 SMC 中，与大鼠颈动脉新生内膜中的半胱天冬酶-3 共定位。
FSS 影响与培养的 SMC 细胞生长和凋亡相关的基因表达，并强烈诱导 TFPI-2 mRNA 和蛋白的表达。TFPI-2 在大鼠颈动脉球囊损伤后的管腔、FSS 暴露的 SMC 中与半胱天冬酶-3 一起表达。功能上，TFPI-2 可能通过调节 SMC 增殖和存活在血管壁修复中发挥作用。需要进一步研究阐明 TFPI-2 控制 SMC 功能的机制。
