Diabetes is associated with augmentation of prothrombogenic von Willebrand factor (vWF) content in plasma. Earlier, the author and colleagues have shown that high glucose and insulin do not appreciably influence deposition of vWF into the subendothelial extracellular matrix (SECM) produced by cultured human umbilical vein endothelial cells (HUVECs). In the present work, the author used this model to test the effects of nonenzymatically glycated albumin (Glyc-HSA) and two lectins, concanavalin A (ConA) and wheat germ agglutinin (WGA), on vWF deposition into the SECM. First-passage HUVECs were seeded into gelatin-coated 96-well plates and cultured for 6 to 7 days. HSA or Glyc-HSA (at concentrations 25, 50, and 100 microg/mL), and WGA or ConA (4, 8, and 16 microg/mL) were added 3 h after seeding. Cell viability was tested by the MTT method. To determine vWF contents in the SECM, HUVECs were detached by treatment with NH4OH and the residual material was used as a solid phase in an enzyme-linked immunosorbent assay (ELISA)-like assay with primary (anti-vWF) and secondary (peroxidase-conjugated) antibodies. Addition of Glyc-HSA did not essentially influence VWF contents in the SECM (A490 was 0.226 versus 0.268 at 0 and 100 microg/mL, respectively; p > .05, n = 16). Cultivation in the presence of WGA led to the deterioration of cell viability, which was accompanied by a significant decrease of vWF in the SECM (0.248 versus 0.128 at 0 and 16 microg/mL, respectively; p < .001, n = 16). ConA did not influence viability of HUVECs, but this lectin at all concentrations consistently increased the deposition of vWF (up to 164% relative to control, p <.001; n = 16). These data indicate that endothelial carbohydrate determinants and corresponding ligands (namely, mannose-specific lectins) may be involved in the regulation of production and deposition of vWF.

译文

糖尿病与血浆中血栓形成原性血管性假血友病因子 (vWF) 含量增加有关。早些时候,作者和同事已经表明,高糖和胰岛素不会明显影响vWF在培养的人脐静脉内皮细胞 (huvec) 产生的内皮下细胞外基质 (SECM) 中的沉积。在当前工作中,作者使用该模型测试了非酶促糖化白蛋白 (Glyc-HSA) 和两种凝集素,伴刀豆球蛋白A (ConA) 和小麦胚芽凝集素 (WGA) 对vWF沉积到SECM中的影响。将首次通过的huvec接种到明胶涂层的96孔板中,并培养6至7天。接种后3小时加入HSA或Glyc-HSA (浓度为25、50和100微克/毫升) 和WGA或ConA (4、8和16微克/毫升)。通过MTT方法测试细胞活力。为了确定SECM中的vWF含量,通过用NH4OH处理分离HUVECs,并将残留的物质用作一级 (抗-vWF) 和二级 (过氧化物酶结合的) 抗体的酶联免疫吸附测定 (ELISA) 样测定的固相。添加Glyc-HSA基本上不影响SECM中的VWF含量 (分别在0和100微克/毫升时A490为0.226对0.268; p> .05,n = 16)。在WGA存在下的培养导致细胞活力的恶化,其伴随着SECM中vWF的显著降低 (分别在0和16微克/毫升时0.248对0.128; p <.001,n = 16)。ConA不影响huvec的生存能力,但是在所有浓度下,这种凝集素始终增加vWF的沉积 (相对于对照高达164%,p <.001; n = 16)。这些数据表明,内皮碳水化合物决定簇和相应的配体 (即甘露糖特异性凝集素) 可能参与了vWF产生和沉积的调节。

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