of VEGF. However, it has not been established that serum IGF-1 in the absence of leaky vessels causes proliferative disease. Although local production of IGF-1 in the retina appears to play only a minor role compared to the considerably higher levels of IGF-1 in the serum, local expression of other components of the GH/IGF-1 signaling pathway in the retina might have an impact on the response of retinal neovessels to IGF-1. This possibility will be discussed in the next chapter with regard to retinal expression of IGFBP-3 as locally regulating the GH/IGF-1 pathway.

IGFBP-3 AS A REGULATOR OF THE GROWTH-HORMONE/ INSULIN-LIKE GROWTH FACTOR PATHWAY

As outlined above, the growth-hormone/insulin-like growth factor pathway appears to be involved in both the development of PDR as well as ROP. This leads to the questions of how GH/IGF-1 signaling might be regulated both endogenously as well as by putative interventions using pharmacological approaches. The IGF-binding proteins (IGFBPs) have been found to play an important role in this respect by regulating both the actions as well as the bioavailability of IGF-1 [63]. Systemically, the vast majority of IGF-1 (up to 98%) is bound to one of the six IGFBPs. Within the IGFBP family, IGFBP-3 is by far the most abundant binding protein, with concentrations in the range of 100 nM, compared with the 2–15-nM concentrations of other binding proteins [64]. IGFBP-3 is bound to IGF-1 or IGF-2 in a ternary complex with a glycoprotein, the acid-labile subunit (ALS). This complexation of IGF-1 with IGFBP-3 and ALS leads to a greatly extended circulating half-life of IGF-1. By increasing IGF’s serum half-life, IGFBP-3 might theoretically increase the biological effects of IGF-1 [65]. Once in the tissue, however, IGFBPs can either potentiate IGF signaling by releasing IGF-1 in proximity of its receptors or, conversely, hinder signaling by sequestering IGF-1 (reviewed in [66]). IGFBP-3 specifically has been found to have mainly inhibitory functions on IGF signaling. IGFBP-3 can inhibit IGF-1 effects by interfering with IGF signaling or by direct, IGF-independent effects (reviewed in [67]). In vitro, addition of IGFBP-3 to HUVECs stimulated with IGF-1 or VEGF reversed both IGF-1- and VEGF-induced proliferation and prevented the survival induced by these factors [68]. IGFBP-3 can directly bind to the retinoid X receptor-alpha independent of IGF. Binding to this receptor can modulate cell cycle and apoptosis through interference with TGF beta and other signaling pathways (reviewed in [66]).

In regard to proliferative retinopathies, IGFBP-3 has been found to be increased in the vitreous of diabetic rats and human diabetic patients. Interestingly, vitreal IGFBPs were elevated even before the onset of overt retinopathy. This was interpreted as vitreal IGFBPs being involved in early ocular events in the diabetic process as opposed to being the result of end-stage retinopathy [69]. Another study measuring serum free and total IGF-1 as well as IGFBP-3 levels in 56 insulin-treated diabetic patients and 52 healthy sexand age-matched controls found lower serum levels both for IGF-1 and IGFBP-3 in diabetic patients. However, age-adjusted free IGF-1 levels in subjects with diabetic retinopathy were higher than those in subjects without diabetic retinopathy [32].

Similar to IGF-1, IGFBP-3 is not only present in the serum but also produced locally in the eye [70]. Retinal expression of IGFBP-3 was found to be highly elevated in rats

that were exposed to hypoxia [71]. Another study investigated the retinal expression of several IGF-linked genes in greater detail using laser-capture microdissection [72]. This study could localize the hypoxia-induced surge in retinal IGFBP-3 to the neovascular tufts suggesting a direct role for IGFBP-3 during the course of proliferative retinopathy. It has not been investigated if IGFBP-3 alters IGF-1 signaling or has a direct, IGF-independent effect in this context. Considering the fact that IGFBP-3 can affect such divergent cellular functions as mobility, adhesion, apoptosis, survival, and the cell cycle, it would be of great interest for future studies to investigate the exact cellular pathways affected by hypoxia-induced local expression of IGFBP-3 in neovascular tufts. Especially in the light of IGFBP-3 having pro-angiogenic effects in some systems while inhibiting it in others [73], it remains open at this point if IGFBP-3 expression in neovascular tufts plays a role in inducing or rather limiting pathologic retinal neovascularization, although lower mRNA expression levels of IGFBP-3 are associated with more retinopathy [75].

In addition to the direct effects of IGFBP-3 on local angiogenesis, recent work from Chang et al. found that IGFBP-3 also has a critical role in promoting migration, tube formation, and differentiation of endothelial progenitor cells (EPCs) [74]. Recruitment of EPCs to neovascular tufts in the hypoxic retina may thus be another possible role for local IGFBP-3 in proliferative retinopathy. At this point it can only be speculated that increased EPC recruitment through retinal IGFBP-3 might lead to a more organized regrowth of normal vessels as opposed to the erratic growth observed in neovascular tuft formation. EPC recruitment might be one of the mechanisms by which IGFBP-3 promotes retinal repair after oxygen-induced vessel loss [75].

THERAPEUTIC CONSIDERATIONS FOR IGFBP-3

IN PROLIFERATIVE RETINOPATHIES

In children with ROP, serum levels of IGF-1 are inversely correlated with disease severity (see Section “Retinopathy of Prematurity (ROP)” of this chapter). Thus, increasing IGF-1 by exogenous administration might appear as a reasonable treatment option to improve ROP risk in premature babies with low IGF-1 levels. However, bolus injections of IGF-1 potentially can induce hypoglycemic episodes [76]. IGF-induced hypoglycemia, however, can be blocked by coadministering equimolar concentrations of IGFBP-3 together with IGF-1. This finding emphasizes the important regulatory role of IGFBP-3 on serum IGF-1 levels and systemic IGF-1 activity. It also stresses the importance of IGFBP-3 for therapeutic interventions involving the GH/IGF-1 pathway in human patients. The importance of IGFBP-3 substitution along with IGF-1 is further stressed by the fact that in premature infants of 30–35 weeks postmenstrual age, IGFBP-3 levels were found to be significantly diminished in infants with ROP compared to those without [75]. Further, in IGFBP-3-deficient mice, there is a dosedependent increase in oxygen-induced retinal vessel loss. Wild-type mice treated with exogenous IGFBP-3 had a significant increase in vessel regrowth without any change in IGF-1 levels. This correlated with a 30% increase in EPCs in the retina at postnatal day 15, indicating that IGFBP-3 could be serving as a progenitor cell chemoattractant. These results suggest that IGFBP-3, acting independently of IGF-1, helps to prevent

oxygen-induced vessel loss and to promote vascular regrowth after vascular destruction in vivo in a dose-dependent manner, resulting in less retinal neovascularization [75]. As a consequence, clinical trials aiming at correcting IGF-1 deficiency in premature infants use equimolar combinations of IGF-1 and IGFBP-3 [77].

In regard to diabetic retinopathy, there is a substantial body of work indicating that hyperglycemia is associated with reduced serum IGF-1 concentrations [32, 78]. Similar to ROP, the early stages of diabetic retinopathy are associated with low levels of systemic IGF-1. From a therapeutic point of view, it has been shown in clinical studies that restoring normal IGF-1 levels in insulin-treated patients using combined IGF-1/ IGFBP-3 regimens results in a concomitant reduction in insulin requirement to maintain euglycemia [79, 80].

One other critical event during the course of diabetic retinopathy is an event known as “early worsening” of proliferative retinopathy. This term refers to an acute increase in retinal proliferative disease coinciding with the onset of exogenous insulin administration. This phenomenon is thought to be linked to an insulin-induced stimulation of the GH/IGF-1 axis. A recent case series with poorly controlled type 1 diabetic patients found that after glycemic control was improved by intensified insulin therapy, serum IGF-1 levels acutely increased and PDR progressed with development of macular edema and proliferation of new vessels [81]. Similarly, a prospective study with 103 pregnant women with type 1 diabetes found that progression of retinopathy during pregnancy was significantly associated with a pregnancy-related increase in IGF-1 levels [82].

It appears likely that the above-described increased serum levels of IGF-1 during “early worsening” of PDR are major contributors to increased retinal IGF-1 signaling. First, serum IGF-1 and IGFBP-3 levels are 10–100 times higher than those measured in the vitreous [26]. Second, patients with PDR show a significant positive correlation between serum and vitreous levels of IGF-1 and the increase in vitreous levels of IGF-1, IGF-2, and IGFBP-3 parallels the increase in vitreous of liver-derived serum proteins [25]. This correlation between serum and vitreal levels is likely due to a diseaseassociated increase in leakiness of the blood-retina barrier of patients with PDR [26, 83]. Measuring serum levels of IGF-1 and IGFBP-3 in diabetic patients can therefore give a good indication of the retina’s exposure to these growth factors. From a therapeutic point of view, it can be speculated that exogenously administered IGFBP-3 could blunt the observed surge of serum IGF-1 by complexing free IGF-1 in the serum and thus preventing IGF-1 from accumulating in the retina. However, the safety of IGFBP-3 administration in PDR patients must be carefully evaluated especially in the context of pregnancy-induced IGF-1 increase.

CONCLUSION

The data on GH, IGF-1, and IGFBP-3 summarized in this chapter illustrate the close association of these three molecules with the development of proliferative retinopathies both in the setting of PDR as well as ROP. This chapter also suggests a number of possibilities to intervene medically in the development of retinopathy by targeting the GH/ IGF-1 pathway. IGFBP-3 is one candidate for therapeutic interventions due to its role as a regulator of the GH/IGF-1 pathway. However, it must be emphasized that timing

is critical to any intervention targeting proliferative retinopathies. Inhibition of IGF-1 early after birth in premature babies or during the early phases of diabetic retinopathy might prevent normal blood vessel development or increase the loss of established retinal vasculature. Instead of IGF inhibition, careful supplementation of IGF-1 might be needed during these early phases of retinopathy. In these cases, IGFBP-3 should be used as an adjunct to IGF-1 supplementation to regulate the bioavailability and activity of exogenously administered IGF-1 and to avoid IGF-induced hypoglycemic episodes. Once active proliferation in the retina has developed (stage II of ROP or PDR), further supplementation of IGF-1 might be deleterious to the retina. At these stages, IGF-1 acts as a permissive factor for proliferative retinopathy and inhibition of IGF-1 might be needed to limit retinal neovascularization. IGFBP-3 might play a role in this context through its inhibitory role on IGF-1 signaling. However, before IGFBP-3 can be suggested for clinical use during the proliferative stages of ROP or PDR, more work needs to be done deciphering the exact effects of IGFBP-3 both on IGF-1 signaling as well as on IGFBP-3’s direct actions independent of IGF-1.

REFERENCES

1. Aiello LM. Perspectives on diabetic retinopathy. Am J Ophthalmol. 2003;136(1):122–35. 2. Wilkinson-Berka JL, Wraight C, Werther G. The role of growth hormone, insulin-like growth factor and somatostatin in diabetic retinopathy. Curr Med Chem. 2006;13(27):3307–17.

3. Jardeleza MS, Miller JW. Review of anti-VEGF therapy in proliferative diabetic retinopathy. Semin Ophthalmol. 2009;24(2):87–92.

4. Chung EJ et al. Combination of laser photocoagulation and intravitreal bevacizumab (avastin) for aggressive zone I retinopathy of prematurity. Graefes Arch Clin Exp Ophthalmol. 2007;245(11):1727–30.

5. Honda S et al. Acute contraction of the proliferative membrane after an intravitreal injection of bevacizumab for advanced retinopathy of prematurity. Graefes Arch Clin Exp Ophthalmol. 2008;246(7):1061–3.

6. Kusaka S et al. Efficacy of intravitreal injection of bevacizumab for severe retinopathy of prematurity: a pilot study. Br J Ophthalmol. 2008;92(11):1450–5.

7. Lalwani GA et al. Off-label use of intravitreal bevacizumab (avastin) for salvage treatment in progressive threshold retinopathy of prematurity. Retina. 2008;28(3 Suppl):S13–8.

8. Mintz-Hittner HA, Kuffel Jr RR. Intravitreal injection of bevacizumab (avastin) for treatment of stage 3 retinopathy of prematurity in zone I or posterior zone II. Retina. 2008;28(6): 831–8.

9. Quiroz-Mercado H et al. Antiangiogenic therapy with intravitreal bevacizumab for retinopathy of prematurity. Retina. 2008;28(3 Suppl):S19–25.

10.Ferrara N. Vascular endothelial growth factor. Arterioscler Thromb Vasc Biol. 2009;29(6): 789–91.

11.Stahl A et al. Rapamycin reduces VEGF expression in retinal pigment epithelium (RPE) and inhibits RPE-induced sprouting angiogenesis in vitro. FEBS Lett. 2008;582(20):3097–102.

12.Schimek RA. Hypophysectomy for diabetic retinopathy; a preliminary report. AMA Arch Ophthalmol. 1956;56(3):416–25.

13.Alzaid AA et al. The role of growth hormone in the development of diabetic retinopathy. Diabetes Care. 1994;17(6):531–4.

14.Kohner EM et al. Pituitary ablation in the treatment of diabetic retinopathy (a randomized trial). Trans Ophthalmol Soc U K. 1972;92:79–90.

37.Ashton N, Ward B, Serpell G. Role of oxygen in the genesis of retrolental fibroplasia; a preliminary report. Br J Ophthalmol. 1953;37(9):513–20.

38.Ashton N, Ward B, Serpell G. Effect of oxygen on developing retinal vessels with particular reference to the problem of retrolental fibroplasia. Br J Ophthalmol. 1954;38(7): 397–432.

39.Kinsey VE et al. PaO2 levels and retrolental fibroplasia: a report of the cooperative study. Pediatrics. 1977;60(5):655–68.

40.Flynn JT. Acute proliferative retrolental fibroplasia: multivariate risk analysis. Trans Am Ophthalmol Soc. 1983;81:549–91.

41.Lutty GA et al. Proceedings of the third international symposium on retinopathy of prematurity: an update on ROP from the lab to the nursery (November 2003, Anaheim, California). Mol Vis. 2006;12:532–80.

42.Smith LE. Pathogenesis of retinopathy of prematurity. Semin Neonatol. 2003;8(6):469–73.

43.Tasman W et al. Retinopathy of prematurity: the life of a lifetime disease. Am J Ophthalmol. 2006;141(1):167–74.

44.Hellstrom A et al. Reduced retinal vascularization in children with growth hormone deficiency. J Clin Endocrinol Metab. 1999;84(2):795–8.

45.Le Roith D et al. The somatomedin hypothesis: 2001. Endocr Rev. 2001;22(1):53–74.

46.Velloso CP. Regulation of muscle mass by growth hormone and IGF-I. Br J Pharmacol. 2008;154(3):557–68.

47.Langford K, Nicolaides K, Miell JP. Maternal and fetal insulin-like growth factors and their binding proteins in the second and third trimesters of human pregnancy. Hum Reprod. 1998;13(5):1389–93.

48.Lassarre C et al. Serum insulin-like growth factors and insulin-like growth factor binding proteins in the human fetus. Relationships with growth in normal subjects and in subjects with intrauterine growth retardation. Pediatr Res. 1991;29(3):219–25.

49.Reece EA et al. The relation between human fetal growth and fetal blood levels of insulin-like growth factors I and II, their binding proteins, and receptors. Obstet Gynecol. 1994;84(1): 88–95.

50.Chen J, Smith LE. Retinopathy of prematurity. Angiogenesis. 2007;10(2):133–40.

51.Hellstrom A et al. Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: direct correlation with clinical retinopathy of prematurity. Proc Natl Acad Sci USA. 2001;98(10):5804–8.

52.Hellstrom A et al. Postnatal serum insulin-like growth factor I deficiency is associated with retinopathy of prematurity and other complications of premature birth. Pediatrics. 2003;112(5):1016–20.

53.Lofqvist C et al. Postnatal head growth deficit among premature infants parallels retinopathy of prematurity and insulin-like growth factor-1 deficit. Pediatrics. 2006;117(6):1930–8.

54.Smith LE. Pathogenesis of retinopathy of prematurity. Growth Horm IGF Res. 2004; 14(Suppl A):S140–4.

55.Smith LE et al. Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor. Nat Med. 1999;5(12):1390–5.

56.Smith LE et al. Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci. 1994;35(1):101–11.

57.Flower RW. Perinatal ocular physiology and ROP in the experimental animal model. Doc Ophthalmol. 1990;74(3):153–62.

58.Penn JS, Tolman BL, Henry MM. Oxygen-induced retinopathy in the rat: relationship of retinal nonperfusion to subsequent neovascularization. Invest Ophthalmol Vis Sci. 1994;35(9):3429–35.

80.O’Connell T, Clemmons DR. IGF-I/IGF-binding protein-3 combination improves insulin resistance by GH-dependent and independent mechanisms. J Clin Endocrinol Metab. 2002;87(9):4356–60.

81.Chantelau E, Frystyk J. Progression of diabetic retinopathy during improved metabolic control may be treated with reduced insulin dosage and/or somatostatin analogue adminis- tration—a case report. Growth Horm IGF Res. 2005;15(2):130–5.

82.Lauszus FF et al. Increased serum IGF-I during pregnancy is associated with progression of diabetic retinopathy. Diabetes. 2003;52(3):852–6.

83.Spranger J et al. Systemic levels contribute significantly to increased intraocular IGF-I, IGF-II and IGF-BP3 [correction of IFG-BP3] in proliferative diabetic retinopathy. Horm Metab Res. 2000;32(5):196–200.