Kiessling F, Becker D, Ullisch E V, Kübler W, Haller C
Medizinische Universitätsklinik, Abt. III, Heidelberg, Germany.
Basic Res Cardiol. 2000 Aug;95(4):299-307. doi: 10.1007/s003950070049.
The endothelium plays a pivotal role in the rheological regulation of blood flow by the secretion of vasoactive factors. The interaction between shear forces and the endothelium is determined by the mechanical properties of the endothelial cell layer which are associated with intercellular junctions. Cell-cell contacts could therefore modulate the secretion of vasocative factors in response to rheological stimuli. We investigated the relationship between intercellular junctions and the secretion of the vasoconstrictor peptide endothelin and the coagulation co-factor von Willebrand factor (vWF). Human umbilical vein endothelial cells (HUVECs) were used as in vitro endothelial model system. Intercellular junctions were reversibly disrupted by calcium chelation or hypertonic stress; alternatively, the formation of intercellular junctions was inhibited by culturing the cells in suspension or by plating them in the presence of an inhibitory anti-VE-cadherin antibody. The opening of intercellular junctions was verified by assessing transmonolayer electrical resistance (TMR) and immunofluorescence morphology. The concentration of endothelin and vWF was measured in the cell culture supernatants using specific ELISAs. The secretion of endothelin was inhibited by EGTA (5 mM) and stimulated by incubation with tumor necrosis factor alpha (TNFalpha, 40 ng/ml). Treatment with hypertonic medium (glycerol, 1,200 mosmol/l) for 10 minutes opened intercellular junctions and markedly reduced the secretion of endothelin. HUVECs in suspension culture did not secrete endothelin and failed to respond to TNFalpha, but readily resumed these functions upon forming a new monolayer on plastic. The reconstitution of intercellular junctions after suspension culture could be inhibited using a specific anti-VE-cadherin antibody. This antibody, but not a non-specific anti-human-IgG antibody reduced endothelin secretion. The secretion of von Willebrand Factor was less dependent on intercellular junctions. The opening of intercellular junctions did not induce cell death, since the cells continued to exclude trypan blue. The results of this study suggest a novel and potentially pathophysiologically/clinically relevant correlation between intercellular junctions and the secretion of endothelin in endothelial cells.
内皮细胞通过分泌血管活性因子在血流的流变学调节中起关键作用。剪切力与内皮细胞之间的相互作用取决于与细胞间连接相关的内皮细胞层的力学性质。因此，细胞间接触可调节血管活性因子的分泌以响应流变学刺激。我们研究了细胞间连接与血管收缩肽内皮素以及凝血辅助因子血管性血友病因子（vWF）分泌之间的关系。人脐静脉内皮细胞（HUVECs）被用作体外内皮模型系统。细胞间连接通过钙螯合或高渗应激被可逆地破坏；或者，通过在悬浮培养细胞或将其接种在抑制性抗血管内皮钙黏蛋白抗体存在的条件下培养来抑制细胞间连接的形成。通过评估跨膜电阻（TMR）和免疫荧光形态学来验证细胞间连接的开放。使用特异性酶联免疫吸附测定（ELISA）法测量细胞培养上清液中内皮素和vWF的浓度。内皮素的分泌受到乙二醇双四乙酸（EGTA，5 mM）的抑制，并通过与肿瘤坏死因子α（TNFα，40 ng/ml）孵育而受到刺激。用高渗培养基（甘油，1200 mosmol/l）处理10分钟可打开细胞间连接并显著降低内皮素的分泌。悬浮培养的HUVECs不分泌内皮素且对TNFα无反应，但在塑料上形成新的单层后可迅速恢复这些功能。悬浮培养后细胞间连接的重建可使用特异性抗血管内皮钙黏蛋白抗体来抑制。这种抗体而非非特异性抗人IgG抗体可降低内皮素的分泌。血管性血友病因子的分泌对细胞间连接的依赖性较小。细胞间连接的开放并未诱导细胞死亡，因为细胞仍能继续排斥台盼蓝。本研究结果表明，内皮细胞中细胞间连接与内皮素分泌之间存在一种新的且可能在病理生理学/临床方面相关的关联。
