IGF-1R and VEGFR-1 Hammerhead Ribozymes Affect Glucose-induced Tight Junction Protein Modifications in Cultured Human Retinal Endothelial Cells

Abstract

Abstract: : Purpose:Increased expression of growth factors such as insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) have been implicated in blood-retinal barrier (BRB) dysfunction associated with diabetic retinopathy. In endothelial cells, adequate expression of the tight junction protein occludin is required for maintenance of BRB function and expression varies in response to stimuli that affect permeability (Gardner TW 1995; Antonetti DA et al 1998). Previously we designed hammerhead ribozymes that specifically cleave the mRNA for specific growth factor receptors and demonstrated reduced receptor protein and function in vivo (Shaw LC et al. 2001). In this study, we investigated the effect of high glucose on occludin expression in human retinal endothelial cells (HRECs) and evaluated whether transfection with either a IGF-1R or VEGFR-1 ribozyme modulate the effect of glucose on occludin expression. Methods:Hammerhead ribozymes that specifically cleave the IGF-1 and VEGFR-1 mRNAs were developed as described (Fritz JJ et al. 2002). HRECs were grown in normal (5 mM) and high (25 mM) glucose medium for 7days and were transfected with plasmid expressing IGF-IR or VEGF-RI hammerhead ribozymes as previously described (Fritz JJ et al. 2002). The amount and distribution of the tight junction protein occludin was analyzed by immunocytochemistry and Western analysis, respectively. Results:Kinetic parameters for IGF-1R ribozyme (V_max = 7.0±0.3 nM/min, K_m = 47.1±1.7, k_cat =0.47±0.01 min^-1, at 25^oC and 1 mM MgCl₂) and the VEGFR-1 ribozyme (V_max=344.8 ±1.4 nM/min, K_m=6.9±1.1 µM, k _cat= 3.0±1.3 min at 37^oC and 20 mM MgCl₂) demonstrate significant catalytic activity in vitro. HRECs grown in low glucose medium showed positive occludin immunoreactivity and protein, while those grown in high glucose depicted reduced immunoreactivity and protein. Transfection of HRECs with active IGF-1R or VEGFR1 hammerhead ribozymes protected against the downregulation of occludin expression that occurred in the cells transfected with the inactive IGF-IR or VEGF-RI ribozymes. Conclusions:Our results demonstrate that the downregulation of occludin by high glucose may be mediated by the VEGF/IGF-1 axis. IGF-1R and VEGFR-1 ribozymes may be potentially useful in preventing BRB breakdown in diabetic retinopathy.

Keywords: diabetic retinopathy • growth factors/growth factor receptors • retinal degenerations: cell biology