Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: direct correlation with clinical retinopathy of prematurity

Abstract

Retinopathy of prematurity is a blinding disease, initiated by lack of retinal vascular growth after premature birth. We show that lack of insulin-like growth factor I (IGF-I) in knockout mice prevents normal retinal vascular growth, despite the presence of vascular endothelial growth factor, important to vessel development. In vitro, low levels of IGF-I prevent vascular endothelial growth factor-induced activation of protein kinase B (Akt), a kinase critical for endothelial cell survival. Our results from studies in premature infants suggest that if the IGF-I level is sufficient after birth, normal vessel development occurs and retinopathy of prematurity does not develop. When IGF-I is persistently low, vessels cease to grow, maturing avascular retina becomes hypoxic and vascular endothelial growth factor accumulates in the vitreous. As IGF-I increases to a critical level, retinal neovascularization is triggered. These data indicate that serum IGF-I levels in premature infants can predict which infants will develop retinopathy of prematurity and further suggests that early restoration of IGF-I in premature infants to normal levels could prevent this disease.

Figures

Figure 1

Effect of IGF-I inhibition on vascular growth. Flat-mounted whole retina shows that, in IGF-I−/− mice (A), there is less progression of vascular development (bright area) compared with IGF-I+/+ littermate controls (B).

Figure 2

Laser microdissection of retina anterior to growing vessels. (A) VEGF mRNA (pink) is visualized anterior to the growing vessels (green fluorescein) in flat-mounted retina. (B) The area containing VEGF (Inset) was removed by laser microdissection in both IGF-I−/− mice and control IGF-I+/+ retinal cross-sections, and VEGF mRNA was analyzed by qRT-PCR relative to cyclophilin control.

Figure 3

Mean serum IGF-I at matched gestational ages in infants with and without ROP. The mean IGF-I level for infants with ROP (○) and without ROP (●) is shown vs. gestational age. (Bars = SEM.)

Figure 4

After serum starvation to reduce baseline Akt phosphorylation, cells were stimulated with VEGF, IGF-I, or both for the times indicated. Replicate blots were prepared from total cell lysates and stained either with phospho-Akt (serine 473) antibody or antibody that recognizes Akt irrespective of phosphorylation status (total-Akt).

Figure 5

Schematic representation of IGF-I/VEGF control of blood vessel development in ROP. (A) In utero, VEGF is found at the growing front of vessels. IGF-I is sufficient to allow vessel growth. (B) With premature birth, IGF-I is not maintained at in utero levels and vascular growth ceases, despite the presence of VEGF at the growing front of vessels. Both endothelial cell survival (Akt) and proliferation (mitogen-activated protein kinase) pathways are compromised. With low IGF-I and cessation of vessel growth, a demarcation line forms at the vascular front. High oxygen exposure (as occurs in animal models and in some premature infants) may also suppress VEGF, further contributing to inhibition of vessel growth. (C) As the premature infant matures, the developing but nonvascularized retina becomes hypoxic. VEGF increases in retina and vitreous. With maturation, the IGF-I level slowly increases. (D) When the IGF-I level reaches a threshold at ≈34 weeks gestation, with high VEGF levels in the vitreous, endothelial cell survival and proliferation driven by VEGF may proceed. Neovascularization ensues at the demarcation line, growing into the vitreous. If VEGF vitreal levels fall, normal retinal vessel growth can proceed. With normal vascular growth and blood flow, oxygen suppresses VEGF expression, so it will no longer be overproduced. If hypoxia (and elevated levels of VEGF) persists, further neovascularization and fibrosis leading to retinal detachment can occur.