Fig 1: MiR-9 transgenic mice showed endothelial specific miR-9 overexpression and inhibition of EndMT. MiR-9 expression in (A) retinal ECs are considerably higher in M9 mice compared with WT mice, whereas miR-9 expression in (B) retinal non-ECs showed no significant difference between M9 and WT. (C) The endothelial marker CD31 (green arrows) showed strong fluorescence in the capillaries of all nondiabetic retinas, as well as in the capillaries of diabetic M9 retinas, and weak fluorescence in the WT diabetic retinas. The mesenchymal marker α-SMA (orange arrow) showed strong fluorescence throughout the retinas of diabetic WT mice, and little to no fluorescence in the capillaries of the other groups. There was considerable overlap between red and green fluorescence in diabetic WT retinas. (n = 6/group for miR-9 analysis and n = 4/group for imaging; miR-9 expression presented as ratio to U6 snRNA and normalized to WT; *P < 0.05, n.s., not significant; fluorescence images were uniformly adjusted to reduce background noise; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; white bar: 10 µm].
Fig 2: High glucose promoted EndMT and suppressed miR-9 in vitro. Exposure to HG (25 mM for 48 hours) reduced the expressions of endothelial markers (A) PECAM1 and (B) CDH5 and increased the expressions of mesenchymal markers (C) TAGLN and (D) S100A4 in HRECs. HG also reduced expression of (E) miR-9. Exposure to high L-glucose (OSM, 25 mM for 48 hours) produced no significance compared to cells grown in NG (5 mM for 48 hours) (n = 6/group; data presented as ratio to β-actin mRNA or U6 snRNA and normalized to NG; *P < 0.05, n.s., not significant).
Fig 3: MiR-9 mimics prevented high glucose-induced EndMT changes in vitro. HG (25 mM for 48 hours) significantly reduced mRNA expressions of endothelial markers (A) PECAM1 and (B) CDH5 and increased mRNA expressions of mesenchymal markers (C) TAGLN and (D) S100A4 in cells transfected with scrambled RNA (SCR). Protein expressions reflect a similar trend, expression of (E) PECAM1 was decreased and expression of (F) S100A4 was increased in SCR-HG compared with SCR cells cultured under NG (5 mM for 48 hours) conditions. MiR-9 mimic transfection (miR-9) prevented high glucose-induced EndMT changes at both mRNA and protein levels (n = 6/group for mRNA and n = 3/group for protein; data presented as ratio to β-actin mRNA/protein and normalized to NG; *P < 0.05).
Fig 4: MiR-9 prevented diabetes-induced EndMT and diabetes-induced vascular leakage in mouse retinas. Streptozotocin-induced diabetes significantly reduced retinal mRNA expressions of endothelial markers (A) Pecam1 and (B) Cdh5, and increased expressions of mesenchymal markers (C) Acta2 and (D) S100a4 in male WT mice. Such changes were prevented in diabetic M9 mice. Protein expressions of (E) Pecam1 and (F) Acta2 also followed the same pattern. (G) IgG staining was seen within the retinal capillaries of all mice (arrow). Diabetes caused leakage of IgG into the retinal layers of WT diabetic mice, resulting in intense (+++) staining throughout the retina when compared with the nondiabetic mice (+). Diabetic M9 mice showed minimal leakage of IgG into the retina and had staining intensity comparable to non-diabetic M9 mice (+). ND, nondiabetic; D, diabetic; M9, miR-9 transgenic (n = 6/group for mRNA, n = 3/group for protein, and n = 3/group for immunohistochemistry; molecular data normalized to WT-ND, mRNA data presented as ratio to β-actin mRNA, and protein data presented as ratio to total protein; *P < 0.05).
Fig 5: MiR-9 regulates TGF-β and inflammatory signaling to regulate EndMT in HRECs. High glucose (HG, 25mM for 48 hours) induced upregulation of (A) MALAT1, (B) NFKB1 and (C) TGFBR2, these changes were prevented by miR-9 mimic transfection. MiR-9 inhibition caused upregulation of (D) MALAT1, (E) NFKB1, and (F) TGFBR2 under normal glucose (NG, 5 mM for 48 hours) conditions. Inhibition of miR-9 (miR-9-i) under normal glucose conditions produced a glucose-like effect and induced EndMT-like changes such as reduced expression of (G) PECAM1 and increased expression of (H) TAGLN (n = 6/group; data presented as ratio to β-actin mRNA and normalized to SCR NG; *P < 0.05).
Supplier Page from Abcam for Mouse CD31 ELISA Kit (PECAM1)