Figure 2.

Conditional depletion of Twist1 in Tie2-expressing cells suppresses pathological neovessel formation in OIR. (A) Schematic illustration shows generation of Tie2-specific conditional Twist1 knockout mice (Tie2-Twist1^cko) created by crossing Tie2-Cre mice with Twist1^flox/flox mice exhibiting disruption in the Twist1 gene in Tie2-expressing cell with active Cre recombinase, which deletes floxed Twist1 gene. (B) Retinal vasculature of Tie2-Cre reporter mice crossed with Rosa mT/mG reporter strain, which universally expresses a red-fluorescent protein (mT). When crossed with Tie2-Cre mice, mT (red) is removed in Tie2-expressing cells with Cre recombinase expression, converting to a green fluorescent protein (mGFP), visible in blood vessels, confirming the expression of Tie-2-driven Cre recombinase in retinal vessels (scale bar: 200 μm). (C) RT-qPCR confirms that Twist1 expression was significantly depleted in OIR retinas of Tie2-Twist1^cko mice compared with Twist1^flox/flox littermate controls at P17 (*P ≤ 0.05). (D) Retinal blood vessel development in Tie2-Twist1^cKO mice and Twist1^flox/flox littermate controls was analyzed at P5 and P7. Representative P7 retinal images with Isolectin B₄ staining (red) are shown for visualization of retinal vasculature (highlighted with yellow dashed line) normalized against total retinal area (white dashed line) (n = 5–11/group at P5, P = 0.59 and n = 8–15/group, P = 0.56, scale bar: 1000 μm). (E) Tie2-Twist1^cKO mice and littermate Twist1^flox/flox control mice were exposed to OIR to induce retinopathy followed by retinal dissection and staining with Isolectin B₄ (red). Tie2-Twist1^cKO retinas show significant suppression of pathological neovessels compared with Twist1^flox/flox controls at P17 in OIR (n = 10–12/group, scale bar: 1000 μm, **P ≤ 0.01). There is no significant difference in vaso-obliteration between the two groups at P17 (n = 10–12/group, P = 0.236; n.s., not significant).