Cell adhesion molecule expression in cultured human iris endothelial cells.
Cell adhesion molecule expression in cultured human iris endothelial cells.
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DOI：
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发表时间：
2001-11
影响因子：
4.4
通讯作者：
M. Silverman;D. Zamora;Y. Pan;P. Texeira;S. Planck;J. Rosenbaum
中科院分区：
文献类型：
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作者：
M. Silverman;D. Zamora;Y. Pan;P. Texeira;S. Planck;J. Rosenbaum
目的开发一种分离人虹膜微血管内皮细胞（HIEC）的方法，以探索其组成型和炎症剂调节的细胞间粘附分子（ICAM）-1和-2、血管细胞粘附分子（VCAM）-1和E-选择素的表达。方法 使用抗体偶联磁珠，根据血小板内皮细胞粘附分子 (PECAM)-1 的表达，从胶原酶消化的虹膜中分离内皮细胞。根据形态学标准、PECAM-1 和冯维勒布兰德因子的表达、乙酰化低密度脂蛋白的摄取以及在合成基底膜上形成毛细血管样网络的能力，将细胞定性为内皮细胞。通过逆转录聚合酶链式反应、酶联免疫细胞测定 (ELICA)、蛋白质印迹分析以及白细胞粘附到 HIEC 单层的功能研究来评估 ICAM-1 和 -2、VCAM-1 和 E-选择素的组成型和炎症剂调节表达。结果 HIEC 持续表达 ICAM-1 和 -2 的 mRNA 和蛋白质，但 VCAM-1 或 E-选择素的水平低至不可检测。当用内毒素或肿瘤坏死因子 (TNF)-α 刺激时，ICAM-1、VCAM-1 和 E-选择素在信息和蛋白质水平上均呈时间和剂量依赖性上调。相比之下，ICAM-2 信息和蛋白质随着时间的推移被炎症因子缓慢下调，但仍然存在并发挥功能。总体而言，HIEC 的细胞因子或内毒素激活导致白细胞的粘附性增强。结论 ICAM-1、VCAM-1 和 E-选择素先前已被认为与介导前眼部炎症有关。这是选择性分离 HIEC 的报告，展示了其中这些粘附分子的差异表达和调节。此外，这是首次证明 ICAM-2 在任何眼部微血管细胞中的表达受到调节。
PURPOSE To develop a method to isolate human iris microvascular endothelial cells (HIECs) for exploring their constitutive and inflammatory agent-modulated expression of intercellular adhesion molecules (ICAM)-1 and -2, vascular cell adhesion molecule (VCAM)-1, and E-selectin. METHODS Endothelial cells from collagenase-digested irises were isolated on the basis of their expression of platelet endothelial cell adhesion molecule (PECAM)-1, using antibody-coupled magnetic beads. Cells were characterized as endothelial based on morphologic criteria, their expression of PECAM-1 and von Willebrand factor, their uptake of acetylated low-density lipoprotein, and their ability to form capillary-like networks on a synthetic basement membrane. Constitutive and inflammatory agent-modulated expression of ICAM-1 and -2, VCAM-1, and E-selectin was evaluated by the reverse transcription-polymerase chain reaction, enzyme-linked immunocellular assays (ELICAs), Western blot analysis, and functional studies of leukocyte adhesion to HIEC monolayers. RESULTS HIECs constitutively expressed mRNA and protein for ICAM-1 and -2, but only low to nondetectable levels of VCAM-1 or E-selectin. When stimulated with endotoxin- or tumor necrosis factor (TNF)-alpha, ICAM-1, VCAM-1, and E-selectin were potently and time- and dose-dependently upregulated at both the message and protein levels. By contrast, ICAM-2 message and protein were slowly downregulated by inflammatory agents over time, but nonetheless remained present and functional. Overall, cytokine- or endotoxin-activation of HIECs resulted in enhanced adhesiveness for leukocytes. CONCLUSIONS ICAM-1, VCAM-1, and E-selectin have been previously implicated in mediating anterior ocular inflammation. This is a report of the selective isolation of HIECs, with a demonstration of differential expression and regulation of these adhesion molecules in them. In addition, this is the first demonstration of the regulated expression of ICAM-2 in any ocular microvascular cells.