Fig. 6.
The cPWWP2A–miR-579–angiopoietin 1/occludin/SIRT1 network regulates retinal vascular dysfunction in vivo. (A and B) Diabetic C57BL/6 mice (2 mo old, male) received an intravitreous injection of miR-579 agomir, negative control (NC) agomir, cPWWP2A shRNA, scrambled (Scr) shRNA, or left untreated (Ctrl) for 1 mo. qRT-PCRs were conducted to detect angiopoietin 1, occludin, and SIRT1 expression (n = 4, *P < 0.05 versus Ctrl group). (C–I) Diabetic C57BL/6 mice (2 mo old, male) received an intravitreous injection of miR-579 agomir, NC agomir, or left untreated (DR) once monthly for a total of 4 mo. “DR” indicates diabetic mice without virus injection. Flat-mounted retinas were double-stained with IB4 and NG2 to visualize pericytes covering retinal vasculature. Tile-scanning images of a leaf of retinal vasculature were taken using a 10× lens. The composite images were constructed by arraying the individual images in Photoshop. The representative composite images and statistical result are shown. (Scale bar in C and D, 200 μm.) Retinal trypsin digestion was performed to detect the number of acellular vascular, pericyte ghost, and microaneurysms (E–G). They were infused with Evans blue dye for 2 h. Tile-scanning images of the entire retinas were taken using a 4× lens. The representative composite images of flat-mounted retinas along with the quantification results of Evans blue leakage are shown. The red fluorescent is Evans blue signaling (H and I, n = 5). (Scale bar, 500 μm.) The significant difference was evaluated by Kruskal–Wallis’s test followed by post hoc Bonferroni’s test.