Insulin-like growth factors promote vasculogenesis in embryonic stem cells

Abstract

The ability of embryonic stem cells to differentiate into endothelium and form functional blood vessels has been well established and can potentially be harnessed for therapeutic angiogenesis. However, after almost two decades of investigation in this field, limited knowledge exists for directing endothelial differentiation. A better understanding of the cellular mechanisms regulating vasculogenesis is required for the development of embryonic stem cell-based models and therapies. In this study, we elucidated the mechanistic role of insulin-like growth factors (IGF1 and 2) and IGF receptors (IGFR1 and 2) in endothelial differentiation using an embryonic stem cell embryoid body model. Both IGF1 or IGF2 predisposed embryonic stem to differentiate towards a mesodermal lineage, the endothelial precursor germ layer, as well as increased the generation of significantly more endothelial cells at later stages. Inhibition of IGFR1 signaling using neutralizing antibody or a pharmacological inhibitor, picropodophyllin, significantly reduced IGF-induced mesoderm and endothelial precursor cell formation. We confirmed that IGF-IGFR1 signaling stabilizes HIF1α and leads to up-regulation of VEGF during vasculogenesis in embryoid bodies. Understanding the mechanisms that are critical for vasculogenesis in various models will bring us one step closer to enabling cell based therapies for neovascularization.

Figure 1. Expression of Insulin Like Growth Factors, Receptors, and Binding Proteins with Embryonic Stem Cell Differentiation.

A) mRNA levels of IGFR-1, IGFR-2, IGF1, and IGF2 increase with time in differentiating embryoid bodies measured from days 1–7, although IGF1 appears to remain steady, pointing to its role in both pluripotency and differentiation. * denotes P<0.05 to day 1 control, Rn denotes normalized reporter.

Figure 2. IGF1 and IGF2 promote mesoderm and endothelial differentiation.

A–B) Day 3 EB treated with IGF1 and IGF2 display increased mRNA levels of Brachyury and no significant change in Oct4, Nanog, and Sox2. C–D) Levels of AFP and PAX6 are not affected. # denotes P<0.05, Rn denotes normalized reporter. Day 7 EB treated with IGF1 (E,F) and IGF2 (G,H) possess increased levels of endothelial specific markers.

Figure 3. Inhibition of IGFR1, but not IGFR2, inhibits vasculogenesis in differentiating embryoid bodies.

A) Tie-2 expression in is decreased in EB treated with IGFR1 neutralizing antibody (nAb IGFR1). Tie-2 expression (FITC), DAPI nuclear counterstain. B) IGFR1 neutralizing antibody treatment (Antibody/Ab) significantly reduces levels of endothelial markers. C) IGFR2 neutralizing antibody treatment had no significant effects on levels of VEGFR2 or Tie-2. * denotes P<0.05 to control, Rn denotes normalized reporter.

Figure 4. Picropodophyllin treatment decreases IGFR1 signaling and downstream AKT and ERK signaling.

A) IGFR1 Y1136 Phosphorylation was inhibited with picropodophyllin (PPP). A random pharmacologic control drug, PHA, had no effect on IGFR1 phosphorylation. B) PPP treatment significantly decreases downstream AKT S473 phosphorylation and ERK p44/p42 phosphorylation (C). * denotes P<0.05 compared to control. (n = 3).

Figure 5. Picropodophyllin treatment inhibits mesoderm and endothelial formation in differentiating embryoid bodies.

Day 3 EB treated with PPP have unchanged or increased levels of pluripotency markers significantly decreased levels of Brachyury. B) PPP treatment decreases expression of vascular markers at day 7. C) Differentiated EB cultures treated with PPP and stained for CD31 (TRITC) had strikingly less vessels compared to control on day 10. *denotes P<0.05, Rn denotes normalized reporter.

Figure 6. IGFs induce endothelial differentiation at early and late stages of vasculogenesis and increase expression of VEGF.

A–B) VEGFR2 and Tie-2 levels at day 7 are significantly increased in EB treated on days 1 and 2 only or on days 4–7 only. C) Treatment with IGF1 or IGF2 post-mesoderm development results in an increase in the percentage of VWF-positive cells compared to control differentiation EB. D–E) PPP treatment on days 1 and 2 only or on days 4–7 only reduced levels of endothelial markers at day 7. Levels of VEGF increased with IGF1 and IGF2 treatment, and decreased with PPP treatment (F–H), at day 3. * denotes P<0.05 compared to control ** P<0.01, Rn denotes normalized reporter.

Figure 7. IGF1 increases HIF1α protein levels in differentiating embryoid bodies and inhibition of HIF1α with rapamycin decreases VEGF and VEGFR2 expression in embryoid bodies treated with IGF1.

A) HIF1αlevels increase significantly following treatment with IGF1. Rapamycin reduces protein levels when added to cell cultures prior to IGF1 stimulation. * denotes P<0.05 compared to conrol, ** denotes P<0.05 compared to +IGF B) Day 3 EB treated with IGF1 express significantly higher levels of VEGF and VEGFR2 compared with control (# denotes P<0.05 compared with control), which are significantly inhibited by treatment with HIF1α inhibitor rapamycin. (* denotes P<0.05 compared to IGF1 treatment). C) Proposed mechanism of IGF1 and IGF2 stimulated endothelial differentiation.