Quantitative image analysis of F-actin in endothelial cells

Objective: Filamentous actin (F-actin) plays a central role in maintaining endothelial barrier function. Thrombin and histamine, two inflammatory medi ators that increase endothelial permeability, can alter F-actin production and distribution. In this study, we use a newly developed image analysis te chnique to show that these two inflammatory mediators differentially alter F-actin structure. Methods: Human umbilical vein endothelial cells were grown to confluence an d treated with either histamine (1 mu-m), thrombin (1 mu-m) 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-acti n image analysis program (FAAP) and converted to layers, each one-pixel thi ck. The program then determined the mean grey 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). Results: Both inflammatory mediators increased endothelial F-actin producti on, however, the distribution of the actin was different. Thrombin increase d the presence of stress fibers. Conclusions: These results establish that thrombin and histamine alter endo thelial F-actin production in different locations within the cell, which ca n be quantified using an image analysis program.