Twist1 Regulates Pathologic Neovascularization in Retinopathy

Abstract

Purpose: Retinal neovascularization (NV) is the blinding pathologic process in proliferative retinopathies such as retinopathy of prematurity and diabetic retinopathy. Twist1, a basic helix-loop-helix domain-containing transcription factor, plays an important role in epithelial-mesenchymal transition and controls tumor angiogenesis. The present study sought to investigate the potential pathogenic role of Twist1 in retinal NV in a mouse model of proliferative retinopathy.

Methods: Conditional depletion of Twist1 expression in mouse vascular endothelial (EC) was developed by using a murine Cre/Lox system. Neonatal Twist1 conditional knockout mice (Tie2-Twist1^cKO) and littermate controls (Twist1^flox/flox) were exposed to 75% oxygen from postnatal (P) day 7-12 and returned to room air from P12-17 in an oxygen-induced retinopathy (OIR) modeling proliferative retinopathy. At P17, when maximum retinal neovascularization was induced, mice were sacrificed and retinas were harvested for analysis with PT-qPCR, Western-blot and immunohistochemical staining. Retinal vascular growth during normal development was also evaluated in Twist1 deficient mice at P7.

Results: Twist1 mRNA expression increased significantly in retinas of OIR mice particularly at P17 (up to 51-fold, p<0.01) compared with room air control mice. Protein levels of Twist1 in OIR retinas were also increased significantly at P17 compared with normoxic controls. Immunohistochemical staining of OIR retinas showed that Twist1 was localized specifically in pathologic retinal NV. Enrichment of Twist1 expression in pathologic NV was confirmed by RT-qPCR analysis of Twist1 mRNA in laser-capture microdissected pathologic NV from OIR retinas compared with normal vessels isolated from normoxic retinas (6-fold increase, p<0.001). Moreover, conditional knockout of Twist1 in vascular endothelial cells resulted in significant suppression of pathological retinal NV areas in OIR at P17 (12.0±0.37% in Tie2-Twist1^cKO vs. 14.83±0.81% in Twist1^flox/flox; p=0.001; n=10-12). At P7, the retinas of Tie2-Twist1^cKO mice showed comparable vascularized areas as littermate controls.

Conclusions: Twist1 is a new specific marker for retinal neovascularization, and regulates pathological vessel formation in proliferative retinopathy. Since Twist1 is dispensable for normal retinal vascular development, it may be a potential therapeutic target for developing treatment for proliferative retinopathy.

Keywords: 700 retinal neovascularization • 706 retinopathy of prematurity • 408 adaptation: motion