Department of General, Visceral, Vascular and Paediatric Surgery (Department of Surgery I), University of Wuerzburg, Oberduerrbacherstraße 6, Wuerzburg D-97080, Germany.
Institute of Anatomy and Cell Biology, University of Munich, Munich D-80336, Germany.
Cardiovasc Res. 2015 Jul 1;107(1):32-44. doi: 10.1093/cvr/cvv144. Epub 2015 May 14.
Microvascular endothelial barrier breakdown in sepsis precedes organ failure and death in patients. We tested the hypothesis that the formation of endothelium-derived soluble vascular endothelial (VE)-cadherin fragments (sVE-cadherin) is involved in inflammation-induced endothelial barrier disruption.
Incubation of human dermal microvascular endothelial cells (HDMEC) with tumour necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (LPS) led to endothelial barrier disruption which correlated with significantly increased sVE-cadherin at a size of ∼90 kDa in cell culture supernatants. Inhibition of the VE-cadherin-cleaving disintegrin and metalloproteinase ADAM10 using GI254023X attenuated inflammation-induced formation of sVE-cadherin and endothelial barrier disruption, suggesting ADAM10-mediated shedding as a mechanism underlying sVE-cadherin release. Formation of VE-cadherin fragments at 90 and 110 kDa was observed when recombinant VE-cadherin (rVE-cadherin) was digested with recombinant ADAM10. Mass spectrometry of the VE-cadherin fragments showed that they originated from cleavage of the extracelluar domain and thereby several cleavage sites of ADAM10 were identified. Atomic force microscopy measurements demonstrated that cell culture supernatants containing sVE-cadherin and application of rVE-cadherin blocked VE-cadherin binding. Accordingly rVE-cadherin dose-dependently led to loss of endothelial barrier functions in HDMEC monolayers. Finally, in patients suffering from severe sepsis or septic shock with clinical signs of a microvascular leackage, serum levels of sVE-cadherin were significantly increased.
Taken together, formation of sVE-cadherin is associated and contributes to inflammation-induced breakdown of endothelial barrier functions by inhibition of VE-cadherin binding. The underlying mechanism of VE-cadherin cleavage involves ADAM10 and appears to be of clinical relevance since sVE-cadherin was augmented in patients with severe sepsis.
脓毒症患者微血管内皮屏障的破坏先于器官衰竭和死亡。我们检验了这样一个假设，即内皮细胞来源的可溶性血管内皮（VE）-钙黏蛋白片段（sVE-cadherin）的形成与炎症诱导的内皮屏障破坏有关。
用肿瘤坏死因子-α（TNF-α）和细菌脂多糖（LPS）孵育人真皮微血管内皮细胞（HDMEC），导致内皮屏障破坏，这与细胞培养上清液中大小约为 90 kDa 的 sVE-cadherin 显著增加相关。使用 GI254023X 抑制 VE-cadherin 切割解整合素金属蛋白酶 ADAM10，可减弱炎症诱导的 sVE-cadherin 形成和内皮屏障破坏，表明 ADAM10 介导的脱落是 sVE-cadherin 释放的一种机制。当重组 VE-cadherin（rVE-cadherin）被重组 ADAM10 消化时，观察到 90 和 110 kDa 的 VE-cadherin 片段形成。VE-cadherin 片段的质谱分析表明，它们来源于细胞外结构域的切割，从而鉴定了 ADAM10 的几个切割位点。原子力显微镜测量表明，含有 sVE-cadherin 的细胞培养上清液和 rVE-cadherin 的应用阻断了 VE-cadherin 的结合。因此，rVE-cadherin 剂量依赖性地导致 HDMEC 单层内皮屏障功能丧失。最后，在患有严重脓毒症或脓毒性休克并有微血管渗漏临床迹象的患者中，sVE-cadherin 的血清水平显著升高。
总之，sVE-cadherin 的形成与炎症诱导的内皮屏障功能破坏有关，并通过抑制 VE-cadherin 的结合而发挥作用。VE-cadherin 切割的潜在机制涉及 ADAM10，并且似乎具有临床相关性，因为严重脓毒症患者的 sVE-cadherin 增加。
