AIMS The pathogenetic mechanisms underlying unprovoked venous thromboembolism (uVTE) are largely unknown. In this study, we investigated the molecular mechanisms involved in uVTE pathogenesis by using ex vivo expanded endothelial colony-forming cells (ECFCs), which represent a valuable non-invasive tool for the assessment of endothelial function. METHODS AND RESULTS We isolated and expanded ECFCs from the peripheral blood of uVTE patients and observed that these cells underwent earlier senescence and showed lower growth rate compared with ECFCs obtained from healthy donors. Through microarray expression profiling, we demonstrated that 2905 genes were differentially expressed between patients and controls. Among them, the anti-angiogenic cytokine TNF superfamily member 15 (TNFSF15) and its death-receptor TNFRSF25 were up-regulated in uVTE ECFCs, and this finding was validated by RT-qPCR. TNFSF15 up-regulation was confirmed at the protein level in ECFC supernatants, and the in vivo relevance of these findings was further corroborated by demonstrating that also the plasmatic levels of TNFSF15 are increased in uVTE patients. After proving that exogenous TNFSF15 exerts pro-apoptotic and anti-proliferative activity on control ECFCs, we demonstrated through blocking experiments that TNFSF15 up-regulation contributes to impaired survival and proliferation of uVTE ECFCs. CONCLUSION By providing evidence that TNFSF15 impairs ECFC functions crucial to endothelial repair, and that uVTE patients have increased TNFSF15 levels both ex vivo and in vivo, the results of this study suggest that pathologic up-regulation of TNFSF15-TNFRSF25 axis may contribute to uVTE pathogenesis, and may represent the target for novel therapeutic strategies aimed at preventing recurrences in uVTE patients.

中文翻译：

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TNFSF15-TNFRSF25轴的病理性上调维持了原发性静脉血栓栓塞中的内皮功能障碍。

目的原发性静脉血栓栓塞症（uVTE）的致病机制在很大程度上尚不清楚。在这项研究中，我们通过使用离体扩增的内皮细胞集落形成细胞（ECFC），研究了uVTE发病机理的分子机制，这是评估内皮功能的一种有价值的非侵入性工具。方法和结果我们从uVTE患者的外周血中分离并扩增了ECFC，与从健康供体获得的ECFC相比，这些细胞经历了更早的衰老并显示出较低的生长速率。通过微阵列表达谱，我们证明了2905个基因在患者和对照之间差异表达。其中，抗血管生成细胞因子TNF超家族成员15（TNFSF15）及其死亡受体TNFRSF25在uVTE ECFC中上调，RT-qPCR证实了这一发现。在ECFC上清液中的蛋白质水平上证实了TNFSF15的上调，并且通过证明uVTE患者的TNFSF15的血浆水平也增加，进一步证实了这些发现的体内相关性。在证明外源性TNFSF15对对照ECFC发挥促凋亡和抗增殖活性后，我们通过阻断实验证明了TNFSF15的上调有助于uVTE ECFC的存活和增殖。结论通过提供证据证明TNFSF15削弱了ECFC对内皮修复至关重要的功能，并且uVTE患者体内和体外TNFSF15水平均升高，该研究结果表明TNFSF15-TNFRSF25轴的病理上调可能与uVTE发病机理有关。 ，