Research Center of Pharmacology and Experimental Therapeutics,Department of Pharmacology, Ernst-Moritz-Arndt-Universityof Greifswald,Greifswald, Germany.
Eur J Clin Pharmacol. 2012 Jun;68(6):923-35. doi: 10.1007/s00228-011-1209-y.
Purpose Hepatic veno-occlusive disease (HVOD) is one of the major complications following hematopoietic stem cell transplantation (HSCT). Although high-dose busulfan is associated with the development of HVOD, the underlying molecular mechanisms are still unknown.Methods Transcriptional gene regulation by busulfan was profiled using Affymetrix GeneChip® Human Genome U133 Plus 2.0 arrays. Messenger RNA (mRNA) expression of regulated genes was assessed by TaqMan real-time polymerase chain reaction (PCR), and protein expression and secretion was determined by enzyme-linked immunosorbent assay (ELISA)in cell supernatants, lysates, and patient plasma.Results Plasma levels of plasminogen activator inhibitor(PAI)-1 significantly increased 48 h after starting busulfan treatment IV in children preconditioned for HSCT. In vitro,busulfan significantly induced plasminogen activator inhibitor-1 (PAI-1) expression in endothelium-like ECV304 cells in a concentration- and time-dependent manner. Comparative transcriptional profiling of busulfan-treated and control ECV304 cells identified differential expression of genes related to coagulation and fibrinolysis, including tissue factor, tissue factor pathway inhibitor-1, protein S, thrombospondin-1, urokinase receptor, and PAI-1, as well as activin A and transforming growth factor beta 1 (TGF-β1). Ingenuity pathway analysis (IPA) suggested TGF-β1 as a central modulator of gene regulation by busulfan. Consequently, expression of tissue factor, urokinase receptor, and PAI-1 mRNA and PAI-1 protein secretion induced by busulfan were significantly reduced by the activin A/TGF-β1 inhibitor SB 431542 in ECV304 and primary endothelial cells.Conclusions This is the first report that directly relates busulfan exposure to antifibrinolytic activity by PAI-1 and hypercoagulation possibly mediated by members of the TGF-β1 family. This suggests further research to evaluate activin A and TGF-β1 as potential targets for HVOD treatment.
肝静脉闭塞病（HVOD）是造血干细胞移植（HSCT）后主要并发症之一。尽管大剂量白消安与 HVOD 的发生有关，但潜在的分子机制尚不清楚。
使用 Affymetrix GeneChip® Human Genome U133 Plus 2.0 阵列对白消安的转录基因调控进行了分析。采用 TaqMan 实时聚合酶链反应（PCR）评估受调控基因的 mRNA 表达，通过酶联免疫吸附试验（ELISA）测定细胞上清液、裂解物和患者血浆中的蛋白表达和分泌。
HSCT 预处理儿童在开始 IV 白消安治疗 48 小时后，血浆中纤溶酶原激活物抑制剂-1（PAI-1）水平显著升高。在体外，白消安以浓度和时间依赖的方式显著诱导内皮样 ECV304 细胞中纤溶酶原激活物抑制剂-1（PAI-1）的表达。对白消安处理和对照 ECV304 细胞的比较转录谱分析鉴定出与凝血和纤溶相关的基因表达差异，包括组织因子、组织因子途径抑制剂-1、蛋白 S、血栓调节蛋白-1、尿激酶受体和 PAI-1，以及激活素 A 和转化生长因子-β1（TGF-β1）。Ingenuity 通路分析（IPA）表明 TGF-β1 是白消安基因调控的中心调节剂。因此，白消安诱导的 ECV304 和原代内皮细胞中组织因子、尿激酶受体和 PAI-1 mRNA 的表达以及 PAI-1 蛋白的分泌均显著降低。
这是第一个直接将白消安暴露与 PAI-1 的抗纤维蛋白溶解活性以及 TGF-β1 家族成员介导的高凝状态相关联的报道。这表明进一步研究评估激活素 A 和 TGF-β1 作为 HVOD 治疗的潜在靶点。
