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Qiuhua Zhang, Youde Jiang, Jordan J. Toutounchian, C R. Yates, Jena J. Steinle; IGFBP-3 Inhibits Retinal Endothelial Cell Adhesion in High Glucose Conditions. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5402.
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The goal of this study was to investigate whether IGFBP-3 inhibits retinal endothelial cell adhesion associated with hyperglycemia.
Human retinal vascular endothelial cells (REC) were grown in high glucose medium (25 mM) for 3 days. On the 2nd day, they were transfected with an endothelial-cell specific IGFBP-3 plasmid (non-IGF binding) at 1 μg/ml for 24 hrs. Cells were starved and treated with TNFα (10 ng/ml) for 4 hrs. Cell proteins were extracted and analyzed for ICAM-1 expression by ELISA. REC were also plated onto attachment factor-coated slides, grown to 90% confluence in high glucose medium and treated as above. Slides were then mounted in a parallel-plate flow chamber and subjected to a continuous flow of U937 human monocytes (105/ml) at shear stresses of 2 dynes/cm2. TNFα was maintained in the flow medium for the duration of the experiment. For in vivo experiments, male rats were made diabetic with a streptozotocin injection (60 mg/kg). Four treatment groups: control + CMV plasmid, diabetic + CMV plasmid, control + IGFBP-3 plasmid and diabetic + IGFBP-3 plasmid were studied. At 2 months, rats were given at 1 μg/μl IGFBP-3 (non-IGF binding) or CMV plasmid by intravitreal injection. Retinal samples were extracted and analyzed for IGFBP-3 levels and ICAM-1 expression by ELISA.
Overexpression of IGFBP-3 (non-IGF binding) in retinal endothelial cells significantly decreased total levels of ICAM-1 when compared to control TNFα-treated samples (p < 0.05). Significant decreases were obtained in retinal endothelial cell adhesion to monocytes in IGFBP-3 plasmid transfected cells vs. not treated samples in the flow chamber system (p < 0.05). Intravitreal injection with IGFBP-3 plasmid into the diabetic retina showed decreased ICAM-1 levels compared with the control group (p < 0.05).
Data suggest that IGFBP-3 reduces endothelial cell adhesion through decreased ICAM-1 levels in a hyperglycemic environment. This is the first demonstration of the role of IGFBP-3 in inhibition of retinal endothelial cell adhesion. Future studies will investigate the mechanism by which native IGFBP-3 may inhibit retinal endothelial cell adhesion.
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