A model of giant vacuole dynamics in human Schlemm's canal endothelial cells
A model of giant vacuole dynamics in human Schlemm's canal endothelial cells
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DOI：
10.1016/j.exer.2010.11.003
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发表时间：
2011-01-01
影响因子：
3.4
通讯作者：
Overby, Darryl R.
中科院分区：
文献类型：
--
作者：
Pedrigi, Ryan M.;Simon, David;Overby, Darryl R.
穿过施累姆氏管的内壁内皮的房水运输可能涉及通过巨大空泡和孔的流动，但是这些结构如何形成以及它们如何影响眼内压（IOP）的调节的机制还没有很好地理解。在这项研究中，我们开发了一种在人Schlemm管内皮细胞（HSCEC）中形成巨空泡的体外模型，该细胞在基底至顶端方向灌注（即，在受控的压降（2或6 mmHg）下流动穿过体内内壁的方向）。将该系统安装在共聚焦显微镜上进行延时正面成像，并将细胞用钙黄绿素（一种荧光活体染料）染色。在灌注开始时，椭圆形空隙区域出现在均匀染色的细胞质内，三维重建显示这些空隙是细胞的圆顶状外囊，形成巨大的空泡样结构或GVL，再现了真正的巨大空泡的经典“图章环”外观。将压降从2 mmHg增加到6 mmHg增加了GVL高度（14 +/- 4 vs. 21 +/- 7 μ m，p < 0.0001）和内皮导水率（1.15 +/- 0.04 vs.2.11 +/- 0.49 μ l min（-1）mmHg（-1）cm（-2）; p < 0.001），但在分离自不同供体的细胞系之间，GVL对压力的反应存在显著差异。在灌注过程中，观察到GVL在细胞层周围“迁移”和聚集，并且尽管保持相同的压降，但经常塌陷。在人脐静脉和猪主动脉内皮细胞中也观察到GVL形成，表明巨空泡形成不是施累姆氏管细胞的独特性质。然而，在这些其他类型的细胞中。灌注过程中很少观察到GVL“迁移”或收缩，这表明Schlemm管内皮细胞可能更好地适应于承受基底部至心尖部的定向压力梯度。总之，我们已经建立了一个体外模型系统来研究巨空泡的动力学，我们已经证明，这个系统再现巨空泡的形态和行为的关键方面。该模型为研究内皮细胞生物力学在正常眼和青光眼眼内压调节中的作用提供了有希望的机会。(C)2010爱思唯尔有限公司保留所有权利。
Aqueous humour transport across the inner wall endothelium of Schlemm's canal likely involves flow through giant vacuoles and pores, but the mechanics of how these structures form and how they influence the regulation of intraocular pressure (IOP) are not well understood. In this study, we developed an in vitro model of giant vacuole formation in human Schlemm's canal endothelial cells (HSCECs) perfused in the basal-to-apical direction (i.e., the direction that flow crosses the inner wall in vivo) under controlled pressure drops (2 or 6 mmHg). The system was mounted on a confocal microscope for time-lapse en face imaging, and cells were stained with calcein, a fluorescent vital dye. At the onset of perfusion, elliptical void regions appeared within an otherwise uniformly stained cytoplasm, and 3-dimensional reconstructions revealed that these voids were dome-like outpouchings of the cell to form giant vacuole-like structures or GVLs that reproduced the classic "signet ring" appearance of true giant vacuoles. Increasing pressure drop from 2 to 6 mmHg increased GVL height (14 +/- 4 vs. 21 +/- 7 mu m, p < 0.0001) and endothelial hydraulic conductivity (1.15 +/- 0.04 vs. 2.11 +/- 0.49 mu l min(-1) mmHg(-1) cm(-2); p < 0.001), but there was significant variability in the GVL response to pressure between cell lines isolated from different donors. During perfusion, GVLs were observed "migrating" and agglomerating about the cell layer and often collapsed despite maintaining the same pressure drop. GVL formation was also observed in human umbilical vein and porcine aortic endothelial cells, suggesting that giant vacuole formation is not a unique property of Schlemm's canal cells. However, in these other cell types. GVLs were rarely observed "migrating" or contracting during perfusion, suggesting that Schlemm's canal endothelial cells may be better adapted to withstand basal-to-apical directed pressure gradients. In conclusion, we have established an in vitro model system to study giant vacuole dynamics, and we have demonstrated that this system reproduces key aspects of giant vacuole morphology and behaviour. This model offers promising opportunities to investigate the role of endothelial cell biomechanics in the regulation of intraocular pressure in normal and glaucomatous eyes. (C) 2010 Elsevier Ltd. All rights reserved.