Figure 7.

Inhibition of Tmod3 Increases Pathological Neovascularization in OIR

(A–D) Intravitreal injection of siTMOD3 or siNC was performed in OIR mouse eyes at P12, with siTMOD3 injected in one eye and siNC in the contralateral eye. Efficiency of siTMOD3 was verified by qPCR (A), showing an ∼50% reduction of Tmod3 expression after treating with siTMOD3 in the OIR retinas. Expression levels were normalized to Gapdh and to negative controls (n = 4 mice per group). Representative retinal whole mounts stained with IB₄ from P17 OIR mice (B) were used for analysis of retinal pathologic vasculature. Quantification of vaso-obliteration (C) and pathological neovascularization (D) at P17 in OIR showed significantly increased neovascular area with siTMOD3 treatment compared with that in contralateral eyes treated with siNC (D), without a significant difference in vaso-obliteration (C) (n = 7 per group). (E) Retinal Tmod3 expression was analyzed in P17 OIR retinas with intravitreal injection of miR-145 inhibitors into one eye and non-targeting negative controls (Neg Ctrl) into the contralateral eye at P12. Tmod3 expression was significantly increased in OIR retinas treated with miR-145 inhibitors by ∼3-fold. Expression levels were normalized to Gapdh and to negative controls (n = 6 mice per group). (F–H) OIR mouse eyes were intravitreally injected with miR-145 inhibitors combined with siTMOD3 in one eye and siNC in the contralateral eye at P12 after removal from an oxygen chamber. Staining with IB₄ in P17 mouse retinas (F) were used for quantification of vaso-obliteration (G) and neovascularization (H). miR-145 inhibitor combined with siTMOD3 treatment showed a significant increase in neovascular area in OIR retinas compared with contralateral eyes treated with the siNC-miR-145 inhibitor cocktail, but no significant effect on vaso-obliteration (n = 8 per group). Scale bars represent 1 mm (B and F). Data are presented as means ± SEM. *p < 0.05; ***p ≤ 0.001.