Abstract
Previously, we showed that insulin growth factor (IGF)-1 binding protein-3 (IGFBP-3), independent of IGF-1, reduces pathological angiogenesis in a mouse model of the oxygen-induced retinopathy (OIR). The current study evaluates novel endothelium-dependent functions of IGFBP-3 including blood retinal barrier (BRB) integrity and vasorelaxation. To evaluate vascular barrier function, either plasmid expressing IGFBP-3 under the regulation of an endothelial-specific promoter or a control plasmid was injected into the vitreous humor of mouse pups (P1) and compared to the non-injected eyes of the same pups undergoing standard OIR protocol. Prior to sacrifice, the mice were given an injection of horseradish peroxidase (HRP). IGFBP-3 plasmid-injected eyes displayed near-normal vessel morphology and enhanced vascular barrier function. Further, in vitro IGFBP-3 protects retinal endothelial cells from VEGF-induced loss of junctional integrity by antagonizing the dissociation of the junctional complexes. To assess the vasodilatory effects of IGFBP-3, rat posterior cerebral arteries were examined in vitro. Intraluminal IGFBP-3 decreased both pressure- and serotonin-induced constrictions by stimulating nitric oxide (NO) release that were blocked by L-NAME or scavenger receptor-B1 neutralizing antibody (SRB1-Ab). Both wild-type and IGF-1-nonbinding mutant IGFBP-3 (IGFBP-3NB) stimulated eNOS activity/NO release to a similar extent in human microvascular endothelial cells (HMVECs). NO release was neither associated with an increase in intracellular calcium nor decreased by Ca2+/calmodulin-dependent protein kinase II (CamKII) blockade; however, dephosphorylation of eNOS-Thr495 was observed. Phosphatidylinositol 3-kinase (PI3K) activity and Akt-Ser473 phosphorylation were both increased by IGFBP-3 and selectively blocked by the SRB1-Ab or PI3K blocker LY294002. In conclusion, IGFBP-3 mediates protective effects on BRB integrity and mediates robust NO release to stimulate vasorelaxation via activation of SRB1. This response is IGF-1- and calcium-independent, but requires PI3K/Akt activation, suggesting that IGFBP-3 has novel protective effects on retinal and systemic vasculature and may be a therapeutic candidate for ocular complications such as diabetic retinopathy.
Highlights
Hepatic insulin-like growth factors (IGF-1 and insulin growth factor (IGF)-2) circulate almost entirely (.99%) bound to binding proteins (IGFBPs), of which there are six
We showed that 1) IGFBP-3 stimulated eNOS activity and is associated with enhanced dephosphorylation of eNOSThr495; 2) nitric oxide (NO) release is IGF-1 independent, but not associated with an increase in intracellular calcium or decreased by blockade of Ca2+/calmodulin-dependent protein kinase II (CamKII); and 3) IGFBP-3 induced NO release was associated with an increase in phosphatidylinositol 3-kinase (PI3K) activity, Akt-Ser473 phosphorylation and selectively blocked by the scavenger receptor-B1 neutralizing antibody (SRB1-Ab) or Phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002
This was determined in 4 fields of view and expressed as a ratio where the value for a P17 age-matched healthy mouse was used as the denominator, resulting in the age-matched control mouse having a horseradish peroxidase (HRP) leakage index of 1
Summary
Hepatic insulin-like growth factors (IGF-1 and IGF-2) circulate almost entirely (.99%) bound to binding proteins (IGFBPs), of which there are six. IGFBP-3 binds 75 to 90% of circulating IGFs in a large ternary complex that consists of IGFBP-3, the acid-labile subunit (ALS) and IGFs [2]. It has become clear that IGFBPs 1–6 have intrinsic biological activity (i.e., IGF/IGF-1Rindependent actions) in addition to binding of IGFs, sequestering active hormones, and limiting IGF biological activity [5,6]. These intrinsic cellular actions include proliferation, differentiation, migration, angiogenesis, and apoptosis in an IGF/IGF-1 receptor (IGF-1R)-independent manner [1,7,8]
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