Fig. 7.

Dgke KO mice have endothelial defects that improve with PGE₂ supplementation. A and B: representative immunofluorescence microscopy images (A) and quantification by digital image analysis (B) of glomerular CD31 expression in Dgke KO kidneys and WT mice 3 days after treatment with PAN or with PBS as a control. CD31 signal was lower in Dgke KOs compared with controls. Glomeruli are highlighted by dotted lines. Scale bars = 50 μm. Quantification data are expressed as average ± SD (n = 3 mice, 10 optical fields per mouse). C: expression of CD31 in cortexes of Dgke KO and WT mice, 3 days after treatment with PAN or with PBS as a control, measured by quantitative real-time PCR. Values are means ± SE from triplicate samples. P values were calculated by Student's t-test. D: Western blot of human umbilical vein epithelial cell (HUVEC) lysates after short hairpin RNA (shRNA)-mediated silencing of DGKE (sh-Dgke) compared with control cells infected with a green fluorescent protein (GFP)-targeting shRNA (sh-GFP). CD31 is lower in DGKE-silenced HUVECs compared with the controls and increases after 4-h exposure to PGE₂ supplemented medium. E: representative differential interference contrast microscopy images of DGKE knockdown and GFP-targeted control HUVECs immediately after wounding the cell monolayers (0 h) and 6 h after exposure to PGE₂-supplemented medium or to Veh-supplemented medium, as a control. Dashed lines were overlaid to the images to mark the border of the wound. F: quantification by digital image analysis of the distance covered by endothelial cells migrated from the border of the wound 5 h after exposure to PGE₂ supplemented medium, expressed as the ratio between covered distance and total width of the wound. Data from triplicate experiments are expressed as average ± SE. P values were calculated by Student's t-test. G: representative immunofluorescence confocal microscopy images of polyvinyl-acetal sponges implanted subcutaneously in Dgke KO mice (8–12 wk of age, n = 4 sponges per mouse, 4 mice) and of WT littermates injected with PGE₂ or with equal volume of Veh on alternate days for 14 days, probed with an antibody against CD31. Scale bars = 20 μm. H and I: quantification by digital image analysis of the number of positive neovascularized structures, expressed as average of CD3-immunoreactive area of CD31-positive structures (H), or as number of CD31-positive structures per random microscopy field (I). Data were obtained analyzing 10 random images per sponge and are expressed as average ± SD; n = 4 sponges per mouse, with 4 mice per each experimental group. P values were calculated by Student's t-test.