Figure 2

ROCK-1 distribution and blebbing in endothelial cells of diabetic retinal vessels. ROCK1-phalloidin double staining (ROCK-1 in green, phalloidin in red) on: (A) Rat retina, (B) Human retina. Endothelial cell blebbing is identified by actin staining only in GK rat retinal vessels (A,a–b). TEM confirms cytoplasmic protrusions with double layered membrane and normal nuclei in GK rats vessels and not in WS rats (A,b–e). In GK rat vessels, ROCK-1 is distributed at the endothelial cell membrane and surrounds the endoluminal blebs (A,g arrows) that reduce the lumen (A,g, double arrow). 3D representation of ROCK-1 and phalloidin staining clearly show the membrane protrusions co-stained with ROCK-1 and phalloidin (A,h). In vessels from a normal human retina, ROCK-1 co-localizes in the cytoplasma of endothelial cells in retinal vessels (B,a), but is translocated at the membrane and in blebs in endothelial cells from a diabetic human retina (B,b), where intense blebbing can be observed (B,c) narrowing the vessel lumen. Scale bar = 10 µm, for TEM scale bar = 0.5 µm.