Impact of the ROCK inhibitor ripasudil on distribution of claudin-5 in vascular endothelial cells in diabetic retinopathy

Abstract

Purpose : Diabetic macular edema (DME) is caused by vascular hyperpermeability associated with redistribution of claudin-5, a molecule that plays an essential role in tight junction assembly between retinal vascular endothelial cells. Here, we investigated the impact of a ROCK inhibitor, ripasudil, on claudin-5 expression and vascular barrier integrity in diabetic retinopathy by using in vitro and in vivo models.

Methods : In vitro, a brain microvascular endothelial cell line, bEND.3, was used to measure the claudin-5 expression and transendothelial electrical resistance (TEER) under conditions stimulated by the addition of either vascular endothelial growth factor (VEGF) or by vitreous fluid of DME, in the presence or absence of ripasudil. In vivo, Kimba mice, which show a transient overexpression of human VEGF in photoreceptor cells, were used to investigate the effect of ripasudil eye drops, intravitreal injection of bevacizumab (IVB), or the combination of both treatments on claudin-5 expression and on leakage of fluorescein dye from vascular vessels.

Results : In vitro, ROCK activity was increased in endothelial cells by addition of VEGF (p<0.05) as well as by addition of vitreous fluid of DME (p<0.05). Ripasudil did not affect VEGF- or DME vitreous fluid-induced reduction of claudin-5 expression in whole cell lysates. However, ripasudil maintained claudin-5 levels in the membrane fraction and the compound also inhibited a decrease in TEER under conditions stimulated by the addition of VEGF (p<0.01 and p<0.0001) or by addition of vitreous fluid of DME (p<0.05 and p<0.0001). In vivo, Kimba mice showed ROCK activation in the retina (p<0.01). Eye drops of ripasudil and IVB partially restored vascular barrier function, and the combination treatment inhibited the vascular hyperpermeability in an additive manner.

Conclusions : ROCK inhibition by ripasudil attenuated vascular hyperpermeability by maintaining claudin-5 in endothelial membranes. Ripasudil could be a new candidate drug for DME.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.