IGFBP-3 Mediates Functional Revascularization in the Retina by Enhancing Nitric Oxide Production Through Activation of the Scavenger Receptor (SR-B1)

Abstract

Purpose: : In this study we investigate whether IGFBP-3, which we have shown to be vascular protective and to promote proper revascularization (Chang et al., PNAS, 2007), exerts its beneficial vascular effects by enhancing nitric oxide (NO) production. We questioned whether the scavenger receptor, class B, type 1 (SR-B1), also known as the HDL receptor, is the receptor responsible for the effects of IGFBP-3.

Methods: : Vascular repair was assessed in the laser vessel occlusion model in adult C57BL/6J.gfp mice receiving intravitreal injection of an IGFBP-3 plasmid under the control of an endothelial cell specific promoter or a control plasmid. For cell culture studies, NO production was assessed using DAF-FM fluorescence microscopy, in human EPCs (CD34⁺ cells) and microvascular cells were treated with physiological concentrations of recombinant IGFBP-3 (100 ng/ml) and/or HDL (1mg/ml). Cells were treated with the HDL receptor blocking antibody (SRB1-Ab) to determine their effects on NO production. eNOS protein expression in IGFBP-3-treated human CD34⁺ cells was assessed by western blotting. Intact arteriole studies were also preformed to assess if IGFBP-3 promotes NO-mediated vasodilatation.

Results: : Overexpression of IGFBP-3 enhanced functional revascularization in the retina of adult mice. In vitro, IGFBP-3 exposure increased eNOS and NO generation in CD34⁺ EPCs. HDL similarly increased NO generation, as did the combination of IGFBP-3 and HDL (p<0.05) in endothelial cells. Treatment with SRB1-Ab significantly decreased NO release by IGFBP-3 alone (P<0.05), by HDL (p<0.05) and by the combination (p<0.01), suggesting that IGFBP-3 increases NO production via activation of SR-B1, as does HDL. Intact arterial studies demonstrated that IGFBP-3 produced vasodilatation by stimulating endothelial NO generation.

Keywords: nitric oxide • retinal neovascularization • protective mechanisms