Department of Cardiology, Shenyang Northern Hospital, Cardiovascular Research Institute, 83 Wenhua Road, Shenyang, 110016, China.
Mol Biol Rep. 2013 Jun;40(6):3891-900. doi: 10.1007/s11033-012-2373-6. Epub 2013 Apr 12.
Cellular repressor of E1A stimulated genes (CREG) is a novel modulator that maintains the homeostasis of vascular cells. The present study aimed to investigate the effects of CREG on tumor necrosis factor (TNF)-α-mediated inflammatory injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured and CREG overexpressing (VC), knockdown (VS) and mock-transfected (VE) HUVECs were challenged with TNF-α. We demonstrated that TNF-α prompted robust intercellular filamentous actin (F-actin) stress fiber formation as examined by rhodamin-phalloidin staining. Transwell assay and rhodamine B isothiocyanate-dextran staining indicated that TNF-α induced intercellular hyperpermeability of the HUVEC monolayers. These effects were attenuated in VC cells with forced CREG overexpression but significantly potentiated in VS cells with CREG silencing. After TNF-α stimulation, interleukin (IL)-6 and IL-8 secretions in VE cells were markedly increased and inducible nitric oxidase (iNOS) expression substantially elevated, whereas these effects were pronouncedly damped in VC cells. Conversely, in VS cells, the increase in inflammatory markers was substantially potentiated. Immunofluorescence staining demonstrated that nuclear factor κB (NF-κB) slowly and transiently translocated into the nuclei of VC cells upon TNF-α stimulation. However, a more swift and sustained nuclear translocation was observed in VS as compared to VE cells. Corresponding changes in the pattern of its protein expression was also observed. These data suggested that CREG can inhibit NF-κB activation, TNF-α-induced inflammatory responses and the hyperpermeability of endothelial cells, and may therefore represent a potential therapeutic target for pathological vascular injury.
细胞 E1A 刺激基因的转录抑制因子（CREG）是一种新型的调节剂，可维持血管细胞的内环境平衡。本研究旨在探讨 CREG 对肿瘤坏死因子（TNF）-α介导的血管内皮细胞炎症损伤的影响。培养人脐静脉内皮细胞（HUVEC），并采用 TNF-α刺激过表达 CREG（VC）、敲低 CREG（VS）和 mock 转染（VE）的 HUVEC。我们发现，TNF-α刺激可导致细胞间丝状肌动蛋白（F-actin）应力纤维形成，罗丹明-鬼笔环肽染色可观察到这一现象。Transwell 实验和罗丹明 B 异硫氰酸酯-葡聚糖染色表明，TNF-α可诱导 HUVEC 单层细胞间的通透性增加。在过表达 CREG 的 VC 细胞中，这些作用减弱，但在敲低 CREG 的 VS 细胞中，这些作用明显增强。TNF-α刺激后，VE 细胞中白细胞介素（IL）-6 和 IL-8 的分泌明显增加，诱导型一氧化氮合酶（iNOS）的表达显著升高，而在 VC 细胞中，这些作用明显减弱。相反，在 VS 细胞中，炎症标志物的增加明显增强。免疫荧光染色显示，TNF-α刺激后，NF-κB 缓慢且短暂地向 VC 细胞核内转位。然而，在 VS 细胞中，NF-κB 的核转位更快且更持久，而在 VE 细胞中则没有。其蛋白表达模式也发生了相应的变化。这些数据表明，CREG 可抑制 NF-κB 的激活、TNF-α诱导的炎症反应和内皮细胞的高通透性，因此可能成为病理性血管损伤的潜在治疗靶点。
