Insulin resistance predisposes to cardiovascular disease (CVD) by inducing endothelial cell (EC) dysfunction and impairs the capacity for endothelial repair. Additionally, we have discovered that a circulating protein, insulin-like growth factor binding protein-1 (IGFBP-1), is potentially protective in the vasculature by stimulating nitric oxide production and enhancing insulin sensitivity. In cross-sectional studies, low IGFBP-1 levels are associated with diabetes and CVD. In this project, we investigated whether IGFBP-1 can enhance vascular endothelial repair in insulin resistant mice in vivo and examined potential mechanisms in human EC and angiogenic progenitor cells (APCs) in vitro .Methods
Endothelial regeneration following femoral artery wire-injury was quantified after 5 days in mice hemizygous for knockout of the insulin receptor (IRKO) with or without transgenic over-expression of human-IGFBP-1. We quantified APCs and assessed function in IRKO, IRKO*IGFBP-1, IGFBP-1 and Wild-type (WT) litter mate controls. Endothelial cell adhesion was assessed ex-vivo in human tissues by seeding segments of endothelium-denuded human saphenous vein with a sub-confluent density of human coronary artery EC, which were pre-incubated with or without IGFBP-1. The effects of IGFBP-1 on the functional properties of EC in vitro were examined using cell migration and proliferation assays. Mechanisms involved in endothelial repair were also investigated through Western blotting for focal adhesion kinase and RhoA activity and Integrin binding assays.Results
Following wire injury, endothelial regeneration was enhanced in IRKO mice expressing IGFBP-1 compared to IRKO controls (47+/-3% vs. 54+/-2%; P < 0.05, Figure 1. A). This was not explained by altered abundance or function of APCs. Incubation of human EC with IGFBP-1 significantly increased cell-surface expression of α5β1 and αVβ3 integrins and enhanced the ability of EC to adhere and regenerate denuded human vein ex vivo (P < 0.001, Figure 1. B). Insulin resistance was induced in EC in vitro by the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-α) which significantly inhibited EC migration (P < 0.01) and proliferation (P < 0.01). Co-incubation with IGFBP-1 restored the migratory (P < 0.05) and proliferative (P < 0.05) capacity of EC to control levels (Figure 2.A and 2.B). IGFBP-1 induced rapid activation of RhoA in EC and significantly increased phosphorylation of focal adhesion kinase.Conclusions
IGFBP-1 ameliorates insulin-resistance related defects in endothelial regeneration by enhancing endothelial cell migration, proliferation and adhesion through mechanisms involving RhoA, integrins and focal adhesion kinase phosphorylation. Our findings raise the possibility that manipulating IGFBP-1 could be a strategy to enhance endothelial repair in patients with insulin resistance.