Insulin-like Growth Factor Binding Protein-3 (igfbp-3) Enhances Junctional Integrity Via Acid Sphingomyelinase to Maintain the Mouse Blood Retinal Barrier (brb)

Abstract

Purpose: : The blood-retinal barrier (BRB) is determined by a complex interaction between tight and adherens junctions. Previously, we have shown IGFBP-3, a member of the IGF system, has marked vascular protective effects (Kielczewski J 2009) and that acid sphingomyelinase, the central enzyme of the sphingolipid pathway converting sphingomyelin to ceramide, plays an important role in stress-induced retinal vascular damage. In this study, we examined whether modulation of IGFBP-3 could alter blood retinal barrier (BRB) function and if this involved acid sphingomyelinase.

Methods: : BRB integrity was assessed in vitro using bovine retinal endothelial cells (BREC). Both transendothelial resistance (TER) and fluorescent flux were measured in IGFBP-3 (100 ng/ml) treated BREC. In vivo, IGFBP-3 was over-expressed using an endothelial cell-specific promoter or by administration of recombinant IGFBP-3 protein. Retinal vascular permeability was induced by either intravitreal VEGF administration in mice or by retinal ischemia following laser obliteration of mouse retinal vessels. Permeability was assessed using retinal flatmounts stained for FITC conjugated albumin. Immunohistochemical analysis of claudin-5 and VE-cadherin expression was also performed in retinal flatmounts. Acid sphingomyelinase (ASMase) mRNA and activity were measured in mouse retinas.

Results: : When IGFBP-3 was applied to the basolateral surface of BREC, TER was increased and fluorescent flux significantly declined. The converse is true when IGFBP-3 is applied to the apical side of BREC. In vivo, intravitreal injection of IGFBP-3 reduced vascular leakage in VEGF injected mice and preserved claudin-5 and VE-cadherin expression compared to VEGF alone-injected mice, which displayed loss of junctional integrity. In laser injured mice, IGFBP-3 expression significantly reduced vascular permeability compared to untreated animals at four days post laser injury (p<0.05) and this was accompanied by a significant decrease in ASMase mRNA expression and activity (p<0.05).

Conclusions: : IGFBP-3 enhances the BRB by increasing maintenance of adherens/tight junction integrity. These effects of IGFBP-3 were associated with a reduction in acid sphingomyelinase expression and activity.

Keywords: retina • diabetic retinopathy • growth factors/growth factor receptors