背景:志贺毒素(Stx)可以激活炎症信号,导致血管功能障碍和促进促血栓形成组织微环境。Stx可引发肠出血性(儿童)溶血性尿毒综合征(eHUS)的发展,血小板减少症三联症,溶血性贫血,和急性肾损伤,经常需要透析。其他特征可能包括对其他器官的损害,包括胃肠道,胰腺,大脑和心血管系统;死亡发生在2-5%。eHUS是一种血栓性微血管病;因此,在肾小球小动脉和其他受累器官的小动脉中,内皮细胞(EC)损伤和血小板纤维蛋白血栓形成是可能的。为了阐明这种微血管病的机制,我们在人类ECs中检查了血小板粘附蛋白P-选择素和血管性血友病因子(VWF)的调节,随着红细胞转化特异性转录因子(ERG)的下调,ERG是血管生成和巨核细胞发育的关键调节因子。
方法:VWF,P-选择素,使用免疫荧光和蛋白质印迹在人脐内皮细胞(HUVECs)中测定ERG水平。HUVECs用肿瘤坏死因子-α(TNF-α)治疗,Stx-1或两者,与正常对照相比。使用处理的HUVEC在Matrigel上进行毛细血管形态发生,用TNF-α治疗22小时,Stx-1,或两者,或单独用Stx-1或与TNF-α联合治疗4小时22小时。
结果:Stx-1显著降低HUVECs上ERG和VWF的表达,但上调P-选择素表达。单独使用Stx-1或与TNF-α联合使用时,ERG水平降低,在核,核周和细胞质区域。Stx-1减少毛细血管形态发生,而Stx-1-TNF-α联合治疗可进一步降低毛细血管形态发生。
结论:在存在Stx-1或TNF-α或两种治疗的情况下,EC被激活,表达较高水平的P-选择素和较低水平的VWF。我们的发现,进一步,提供证据证明Stx-1下调ERG,抑制体外血管生成。
BACKGROUND: Shiga toxin (Stx) can activate inflammatory signaling, leading to vascular dysfunction and promotion of a pro-thrombotic tissue microenvironment. Stx can trigger the development of the enterohemorrhagic (childhood) hemolytic uremic syndrome (eHUS), a triad of thrombocytopenia, hemolytic anemia, and acute kidney injury, often requiring dialysis. Additional features may include damage to other organs, including the gastrointestinal tract, pancreas, brain and cardiovascular system; death occurs in 2-5 %. eHUS is a thrombotic microangiopathy; thus, endothelial cell (EC) injury and platelet fibrin thrombus formation in glomerular arterioles and in the arterioles of other affected organs are likely. To elucidate mechanisms of this microangiopathy, we examined in human ECs the regulation of the platelet adhesion proteins P-selectin and von Willebrand factor (VWF), along with the downregulation of erythroblast-transformation-specific transcription factor (ERG) a key regulator of angiogenesis and megakaryocyte development.
METHODS: VWF, P-selectin, and ERG levels were determined using immunofluorescence and Western blot in human umbilical endothelial cells (HUVECs). HUVECs were treated with tumor necrosis factor-alpha (TNF-α), Stx-1 or both, versus normal controls. Capillary morphogenesis on Matrigel was performed using HUVECs treated, for 22 h with TNF-α, Stx-1, or both, or treated 4 h with Stx-1 alone or in combination with TNF-α for 22 h.
RESULTS: Stx-1 significantly reduced ERG and VWF expression on HUVECs, but upregulated P-selectin expression. ERG levels decreased with Stx-1 alone or in combination with TNF-α, in the nuclear, perinuclear and cytoplasmatic regions. Stx-1 reduced capillary morphogenesis, while Stx-1-TNF-α combined treatment reduced capillary morphogenesis still further.
CONCLUSIONS: In the presence of Stx-1 or TNF-α or both treatments, ECs were activated, expressing higher levels of P-selectin and lower levels of VWF. Our findings, further, provide evidence that Stx-1 downregulates ERG, repressing angiogenesis in vitro.