Abstract：
Deep vein thrombosis (DVT) is a common complication in hematologic malignancies and immunologic disorders. Endothelial cell injury and dysfunction comprise the critical contributor for the development of DVT. A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), a plasma metalloprotease that cleaves von Willebrand factor, acts as a critical regulator in normal hemostasis. This study was aimed to explore the role of ADAMTS13 in endothelial cell injury during DVT and the possible mechanism. First, human umbilical vein endothelial cells (HUVECs) were exposed to hydrogen peroxide (H2O2). Then, the mRNA and protein expressions of ADAMTS13 were evaluated with the reverse transcription-quantitative polymerase chain reaction and western blot. After treatment with recombinant ADAMTS13 (rADAMTS13; rA13), the viability and apoptosis of H2O2-induced HUVECs were assessed by cell counting kit-8 assay and terminal-deoxynucleoitidyl transferase-mediated nick end labeling staining. In addition, the levels of prostaglandin F1-alpha, endothelin-1, and reactive oxygen species were detected using the enzyme-linked immunosorbent assay and dichloro-dihydro-fluorescein diacetate assay. The expressions of proteins related to p38/extracellular signal-regulated kinase (ERK) signaling pathway were estimated with the western blot. Then, p79350 (p38 agonist) was used to pretreat cells to analyze the regulatory effects of rA13 on p38/ERK signaling in H2O2-induced HUVEC injury. The results revealed that ADAMTS13 expression was significantly downregulated in H2O2-induced HUVECs. The reduced viability and increased apoptosis of HUVECs induced by H2O2 were revived by ADAMTS13. ADAMTS13 also suppressed the oxidative stress in HUVECs after H2O2 treatment. Besides, ADAMTS13 was found to block p38/ERK signaling pathway, and p79350 reversed the impacts of ADAMTS13 on the damage of HUVECs induced by H2O2. To sum up, ADAMTS13 could alleviate H2O2-induced HUVEC injury through the inhibition of p38/ERK signaling pathway.
摘要：
深静脉血栓形成（DVT）是血液系统恶性肿瘤和免疫疾病的常见并发症。内皮细胞损伤和功能障碍是DVT发展的关键因素。具有血小板反应蛋白基序13的解整合素和金属蛋白酶（ADAMTS13），一种切割血管性血友病因子的血浆金属蛋白酶，在正常止血中起着关键的调节作用。本研究旨在探讨ADAMTS13在DVT时内皮细胞损伤中的作用及可能机制。首先,将人脐静脉内皮细胞（HUVECs）暴露于过氧化氢（H2O2）。然后，用逆转录-定量聚合酶链反应和Westernblot检测ADAMTS13的mRNA和蛋白表达。用重组ADAMTS13（rADAMTS13；rA13）治疗后，通过细胞计数试剂盒-8测定和末端脱氧核苷酸转移酶介导的缺口末端标记染色评估H2O2诱导的HUVECs的活力和凋亡。此外,前列腺素F1-α的水平，内皮素-1和活性氧使用酶联免疫吸附测定法和二氯-二氢-荧光素二乙酸酯测定法进行检测。免疫印迹法检测p38/细胞外信号调节激酶(ERK)信号通路相关蛋白的表达。然后，p79350（p38激动剂）用于预处理细胞，以分析rA13对H2O2诱导的HUVEC损伤中p38/ERK信号传导的调节作用。结果表明，在H2O2诱导的HUVEC中，ADAMTS13的表达显着下调。ADAMTS13恢复了H2O2诱导的HUVECs活力降低和凋亡增加。ADAMTS13还抑制H2O2处理后HUVECs的氧化应激。此外,发现ADAMTS13阻断p38/ERK信号通路,p79350逆转了ADAMTS13对H2O2诱导的HUVECs损伤的影响。总而言之,ADAMTS13可通过抑制p38/ERK信号通路减轻H2O2诱导的HUVEC损伤。
