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

糖尿病与血浆中促血栓性血管性血友病因子(vWF)含量的增加有关。早前,作者和同事已经表明,高葡萄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。在目前的工作中,作者使用该模型测试了非酶促糖基化白蛋白(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。将第一代HUVEC接种到明胶包被的96孔板中,并培养6至7天。播种后3小时,添加HSA或Glyc-HSA(浓度分别为25、50和100微克/毫升)和WGA或ConA(4、8和16微克/毫升)。细胞存活力通过MTT方法测试。为了确定SECM中的vWF含量,通过用NH4OH处理将HUVEC分离,并将残留的物质作为固相用于类似酶联免疫吸附测定(ELISA)的类似测定中,该测定具有一级(抗vWF)和二级(过氧化物酶-偶联的)抗体。添加Glyc-HSA基本上不影响SECM中的VWF含量(0和100 microg / mL时A490分别为0.226和0.268; p> .05,n = 16)。在存在WGA的情况下进行培养会导致细胞活力下降,并伴有SECM中的vWF显着下降(0和16 microg / mL分别为0.248和0.128; p <.001,n = 16)。 ConA不会影响HUVEC的生存力,但是该凝集素在所有浓度下均会持续增加vWF的沉积(相对于对照,高达164%,p <.001; n = 16)。这些数据表明内皮糖决定簇和相应的配体(即,甘露糖特异性凝集素)可能参与vWF的产生和沉积的调节。

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