Banz Yara, Cung Trinh, Korchagina Elena Y, Bovin Nicolai V, Haeberli André, Rieben Robert
Department of Clinical Research, University of Bern, Bern, Switzerland.
Xenotransplantation. 2005 Nov;12(6):434-43. doi: 10.1111/j.1399-3089.2005.00239.x.
Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting.
Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads.
Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS.
This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ''endothelial protective'' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.
研究异种反应性抗体、补体和凝血因子在超急性和急性血管排斥反应中与内皮细胞的相互作用通常需要使用体内模型。传统的体外或离体系统需要血清、血浆或抗凝全血，这使得凝血介导效应的分析变得困难。本文描述了一种基于微载体的新型体外系统，用于研究内皮细胞（EC）的活化和损伤，该系统使用非抗凝全血。该模型建立后，用于研究具有特征性的补体和凝血抑制剂硫酸葡聚糖（DXS，分子量5000）在异种移植环境中对内皮细胞的保护特性。
将在微载体珠上生长至汇合的猪主动脉内皮细胞（PAEC）与非抗凝全人血孵育，直至发生凝血或最长孵育90分钟。PAEC珠与DXS进行预孵育或共孵育。磷酸盐缓冲盐水（PBS）实验作为对照。分析补体和凝血的液相和表面活化标志物以及DXS与PAEC珠的结合。
DXS的共孵育和预孵育，随后洗涤珠子，显著延长了凝血时间，从39±12分钟（PBS对照）分别延长至74±23分钟和77±20分钟（与PBS相比，P<0.005）。DXS处理减弱了C1q、C4b/c、C3b/c和C5b-9的表面沉积，而不影响IgG或IgM的沉积。通过血管性血友病因子阳性表示的内皮完整性在DXS处理下维持的时间更长。与PBS对照相比，DXS的预孵育和共孵育均显著延长了活化部分凝血活酶时间（>300秒，P<0.05），并减少了凝血酶-抗凝血酶复合物和纤维蛋白肽A的产生。虽然DXS共孵育完全阻断了经典途径补体活性（CH50试验），但DXS预孵育或PBS对照实验未显示抑制作用。使用荧光素标记的DXS可视化显示DXS与PAEC珠结合。
这种新型的体外微载体模型可用于研究内皮细胞损伤以及与全血的复杂相互作用，并可在异种移植环境中筛选“内皮保护”物质。在这种体外环境中，DXS提供内皮保护，减轻超急性异种移植排斥反应中所见的内皮细胞损伤。
