Nuclear Factor of Activated T Cells Is Activated in the Endothelium of Retinal Microvessels in Diabetic Mice

Figure 2

High glucose activates NFATc3 in the endothelium of retinal vessels. (a) Representative confocal immunofluorescence images of retinal whole-mounts, stimulated for 30 min in low (LG; 2 mmol/L; left panels) or high (HG; 20 mmol/L; right panels) extracellular glucose and stained for NFATc3 (red) and SYTOX green for identification of nuclei (green). Merged and individual channel images are shown. Endothelial cells were identified by the orientation of their nuclei. (b) Higher magnification confocal images of retinal whole-mounts stimulated and stained as in ((a), upper panels) and pseudocolored images to visualize colocalization of NFATc3 and SYTOX green (white; lower panels). Scale bars = 50 μm. (c) Summarized mean fluorescence intensity data from experiments as in (a), showing NFATc3 nuclear accumulation after 30 min stimulation in LG or HG in the presence or absence of A-285222 (1 μmol), or apyrase (3.6 U/mL), or after stimulation with LG plus mannitol (18 mmol/L), or LG plus L-glucose (18 mmol/L). and 22 retinas for LG and HG, respectively; 5–7 retinas for the other stimulatory conditions. versus all other groups. (d) Summarized mean fluorescence intensity data showing NFATc3 nuclear accumulation in mouse cerebral arteries after 30 min stimulation in LG or HG in the presence or absence of A-285222 (1 μmol). /group. versus LG.