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First published online April 24, 2006
doi: 10.1242/10.1242/jcs.02886

Journal of Cell Science 119, 1926-1932 (2006)
Published by The Company of Biologists 2006
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Differential action of steroid hormones on human endothelium

Hans Oberleithner1,*, Christoph Riethmüller1, Thomas Ludwig1, Victor Shahin1, Christian Stock1, Albrecht Schwab1, Martin Hausberg2, Kristina Kusche3 and Hermann Schillers1

1 Institute of Physiology II, University Münster, 48149 Münster, Germany
2 Department of Internal Medicine D, University Münster, 48149 Münster, Germany
3 Institute of Zoophysiology, University Münster, 48149 Münster, Germany


Figure 1
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  		Fig. 1. AFM images of HUVEC monolayers viewed from the top. Cells were exposed for 72 hours to solvent (control), to aldosterone, or to aldosterone and spironolactone. The height bar below quantifies the height of the images.

 

Figure 2
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  		Fig. 2. Apical plasma membrane surface of individual endothelial cells (HUVEC). Cells were exposed for 72 hours to solvent (control), to aldosterone (aldo), to aldosterone and spironolactone (aldo+spiro), to dexamethasone (dexa), or to dexamethasone and RU486 (dexa+RU). Mean values ± s.e.m. are shown; the number of scans is shown in the bars, each scan represents between 7-15 cells. Asterisk indicates significant difference in comparison with control (P<0.01).

 

Figure 3
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  		Fig. 3. Endothelial cell stiffness (elastic modulus) in living HUVEC. Cells were exposed for 72 hours to solvent (control), to aldosterone (aldo), to aldosterone and spironolactone (aldo+spiro), to dexamethasone (dexa), or to dexamethasone and RU486 (dexa+RU). Mean values ± s.e.m. are shown; the number of individual cells in which stiffness was measured is shown in the bars. Asterisk indicates significant difference in comparison with control (P<0.01).

 

Figure 4
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  		Fig. 4. (A) Transendothelial dextran permeability measured with fluorescence-labeled 40 kDa dextran. Cells were exposed for 72 hours to solvent (control), to aldosterone (aldo), to aldosterone and spironolactone (aldo+spiro), to dexamethasone (dexa), or to dexamethasone and RU486 (dexa+RU). Mean values ± s.e.m. are shown; the number of measurements on individual monolayers is shown in the bars. Asterisk indicates significant difference in comparison with control (P<0.01); § indicates significant difference in comparison with dexamethasone (P<0.02). (B) Transendothelial electrical resistance measured with the electrical cell impedance sensing (ECIS) technique. Cell treatment was as described in section A. Mean values ± s.e.m. are shown; the number of measurements on individual monolayers is shown in the bars. Asterisk indicates significant difference in comparison with control (P<0.01).

 

Figure 5
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  		Fig. 5. Identification of {alpha}ENaC in human endothelial cells (HUVEC; representative experiment). Membrane proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue (lane 1). {alpha}ENaC was detected by using an anti-{alpha}ENaC antibody (lanes 2-4). The amount of {alpha}ENaC in the aldosterone-treated sample (A) is about twice that in the control (C) and about 6 times higher than that of aldosterone and spironolactone (A+S)-incubated HUVEC. As positive control (PC), we used ENaC-expressing oocytes. The molecular mass standard is given on the left. The upper band in the range of 90 kDa most likely represents a glycosylated form of ENaC.

 

Figure 6
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  		Fig. 6. Comparison between measurements in human endothelial cells (HUVEC) before and after fixation (paired experiment). (Left) AFM image showing a HUVEC monolayer scanned in vivo in HEPES buffer at 37°C. (Right) AFM image showing the same monolayer after glutaraldehyde fixation. Numbers in images indicate corresponding cells.

 

Figure 7
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  		Fig. 7. Principle of cell stiffness measurements (force-distance curves) performed on living human endothelial cells with an atomic force microscope (AFM). For technical details, see Materials and Methods.

 




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