Figure 3.

Tie2-driven conditional knockout of Twist1 results in suppression of laser-induced choroidal neovascularization. (A) Tie2-Twist1^cKO mice and littermate Twist1^flox/flox controls were subjected to laser photocoagulation to induce CNV. Seven days after treatment, choroids were dissected, stained with Isolectin B₄ (red), and flat-mounted for imaging. (B) Quantification of CNV lesion sizes shows that Tie2-Twist1^cKO lesions are significantly smaller compared with lesions from Twist1^flox/flox littermate controls (n = 20–26 eyes/group, *P ≤ 0.05; ON, optic nerve head; scale bar: 100 μm). (C) CNV leakage in Tie2-Twist1^cKO mice and littermate floxed controls was imaged with fundus fluorescent angiography 6 days after laser treatment. Images were taken at 5 and 10 minutes after intraperitoneal injection of fluorescein sodium. (D) Quantification of fluorescent intensity of exudated fluorescein sodium and leakage grading show significantly decreased levels of vascular leakage in Tie2-Twist1^cKO mice compared with littermate Twist1^flox/flox control. Differences in fluorescent intensity of exudated fluorescein sodium between 5 and 10 minutes were quantified and normalized against that of the Twist1^flox/flox littermate control as an indicator of vascular leakage. In addition, levels of leakage were evaluated by leakage grading according to established methods. Grade 0: not leaky; grade 1: questionable leakage; grade 2a: leaky; grade 2b: pathologically significant leakage (n = 18–27 eyes/group, *P ≤ 0.05, scale bars: 200 μm).