Ehringer W D, Yamany S, Steier K, Farag A, Roisen F J, Dozier A, Miller F N
Center for Applied Microcirculatory Research, University of Louisville, School of Medicine, KY 40292, USA.
Microcirculation. 1999 Dec;6(4):291-303.
Filamentous actin (F-actin) plays a central role in maintaining endothelial barrier function. Thrombin and histamine, two inflammatory mediators that increase endothelial permeability, can alter F-actin production and distribution. In this study, we use a newly developed image analysis technique to show that these two inflammatory mediators differentially alter F-actin structure.
Human umbilical vein endothelial cells were grown to confluence and treated with either histamine (1 microM), thrombin (1 microM) or the agonist's vehicle. The endothelium was stained with BODIPY-phallodin, and digitized images were taken of the treated cells. The digitized images of individual human umbilical vein endothelial cells (HUVEC) were imported into a F-actin image analysis program (FAAP) and converted to layers, each one pixel thick. The program then determined the mean gray level (which corresponded to the amount of F-actin) in each layer starting from the outside of the cell (layer 1) and progressing in one pixel layer increments towards the center of the cell (layer 32).
Both inflammatory mediators increased endothelial F-actin production, however, the distribution of the actin was different. Thrombin increased the presence of stress fibers, while also decreasing peripheral banding actin. In contrast, histamine had no effect on peripheral actin compared to control, but did increase the presence of F-actin stress fibers.
These results establish that thrombin and histamine alter endothelial F-actin production in different locations within the cell, which can be quantified using an image analysis program.
丝状肌动蛋白（F-肌动蛋白）在维持内皮屏障功能中起核心作用。凝血酶和组胺这两种增加内皮通透性的炎症介质可改变F-肌动蛋白的产生和分布。在本研究中，我们使用一种新开发的图像分析技术来表明这两种炎症介质对F-肌动蛋白结构的改变存在差异。
将人脐静脉内皮细胞培养至汇合状态，并用组胺（1微摩尔）、凝血酶（1微摩尔）或激动剂的溶剂进行处理。用BODIPY-鬼笔环肽对内皮进行染色，并拍摄处理后细胞的数字化图像。将单个脐静脉内皮细胞（HUVEC）的数字化图像导入F-肌动蛋白图像分析程序（FAAP）并转换为各厚一个像素的层。然后该程序从细胞外部（第1层）开始，以一个像素层的增量向细胞中心（第32层）推进，确定每层的平均灰度值（对应于F-肌动蛋白的量）。
两种炎症介质均增加了内皮F-肌动蛋白的产生，然而，肌动蛋白的分布不同。凝血酶增加了应力纤维的存在，同时也减少了外周带状肌动蛋白。相比之下，与对照组相比，组胺对外周肌动蛋白没有影响，但确实增加了F-肌动蛋白应力纤维的存在。
这些结果表明，凝血酶和组胺在细胞内不同位置改变内皮F-肌动蛋白的产生，这可以使用图像分析程序进行量化。
