Timothy J. Schoen

Evidence for an insulin-like growth factor autocrine-paracrine system in the retinal photoreceptor-pigment epithelial cell complex

: The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin‐like growth factor I (IGF‐I) immunoreactivity that co‐elutes with authentic human IGF‐I in HPLC analyses. Cultured human RPE cells synthesize and release IGF‐I, raising the possibility that the RPE serves as a source of IPM IGF‐I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF‐I receptors with α‐subunits of 120 and 138 kDa, respectively. They thus appear to be of the “brain” (in photoreceptors) and “peripheral” (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF‐BP) that specifically binds IGF‐I and IGF‐II. The IPM‐BP is visualized as a single radiographic band by both ligand blot and affinity cross‐linking procedures. With enzymes specific for removing N‐and O‐linked oligosaccharides, the IPM‐BP was found to contain O‐but not N‐linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM‐BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF‐I and IGF‐BP in the IPM, together with the presence of IGF‐I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine‐paracrine system.

Vitreal insulin-like growth factor binding proteins (IGFBPs) are increased in human and animal diabetics

Although patients with diabetic retinopathy have been reported to have elevated vitreal IGF-I levels, it is not known whether diabetes also affects the levels of vitreal IGF binding proteins (IGFBPs) which control IGFs bioavailability. To address this issue, vitreal IGFBP levels were assayed in human diabetics, rats with streptozotocin-induced diabetes and galactose-fed dogs with diabetic-like retinopathy. Using 125I-IGF-II ligand blots, it was found that human diabetics have a 4-fold increase in vitreal IGFBP levels. Also, western blots on human diabetic vitreous reveal increased levels of IGFBP-2 and proteolytic fragments of IGFBP-3. IGF binding assays on vitreous from streptozotocin-treated rats (three months in duration) also indicate a 5-fold increase in IGF binding activity. IGF ligand blots using vitreous from rats with a shorter duration of diabetes (one month) show a 63% increase in IGFBP binding and a marked decrease in serum IGFBP binding. IGF ligand blots and IGFBP-2 and -4 western blots using vitreous from galactose-fed dogs with diabetic-like retinopathy exhibit a 6-fold increase in vitreal IGFBPs. The observation that vitreal IGFBPs are elevated in diabetic humans and rats without overt retinopathy suggests that these increases are not the result of a preexisting end-stage retinopathy but rather are an early ocular event in the diabetic process. Increases in vitreal IGFBPs thus could participate in the proliferative aspects of diabetic retinopathy by virtue of their putative intrinsic bioactivity or their capacity to alter IGF bioavailability.

An estrogen receptor repressor induces cataract formation in transgenic mice

Despite the high prevalence of age-related cataracts, there are currently no known therapies to delay or prevent their occurrence. Studies in humans and rodent models suggest that estrogen may provide protection against age-related cataracts. The discovery of ocular estrogen receptors (ERs) indicates that estrogen protection may result from direct interactions with its receptors in the eye, instead an indirect consequence from effects on another tissue. Studies in our transgenic mouse model validate the concept that estrogen is beneficial for the eye. These mice express ERΔ3, a dominant-negative form of ERα that inhibits ERα function. In the ERΔ3 transgenic mice, cortical cataracts spontaneously form in ERΔ3 females after puberty and progress with age. The cataracts initiate in the equatorial region of the lens where the epithelial cells differentiate into elongating fiber cells. Cataract formation can be prevented if the females are ovariectomized before, but not after, sexual maturity. Both male and female ERΔ3 mice develop cataracts after neonatal treatment with the potent estrogen diethylstilbestrol (DES). The incidence of spontaneous and DES-induced cataracts in ERΔ3 mice is 100%, yet these cataracts are absent from the wild-type mice. These data suggest that repression of estrogen action induces cortical cataract formation because estrogen is required to activate the ERΔ3 repressor. Evidence of DES-induced cataracts in the ERΔ3 males as well as the females suggests that estrogen is important in lens physiology in both sexes. The ERΔ3 mice provide a powerful model for assessing the role of estrogen in maintaining the transparency of the lens.

IDENTIFICATION AND PARTIAL CHARACTERIZATION OF A PROTEINASE SPECIFIC FOR INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 IN AQUEOUS AND VITREOUS HUMORS

The IGFs (-I and -II) are normally found in serum and other extracellular fluids complexed to specific binding proteins (IGFBPs). While several IGFBPs have been identified in vitreous and aqueous humors, the major serum carrier of IGF, IGFBP-3, is notably absent from these fluids. To determine if this paucity could be due to an IGFBP-3 proteinase (IGFBP-3ase), samples of bovine vitreous or aqueous humor were mixed with serum and incubated at 37 degrees C for 4 h followed by western ligand blotting. In these experiments, a distinct loss of the 46 kDa band representing IGFBP-3 was observed while other bands present at 35, 28 and 25 kDa were unaltered. The IGFBP-3ase activity is temperature sensitive, has a pH optimum of about 8.0 and is inhibited by EDTA. Acid treatment of serum to remove endogenously bound IGF does not affect the specificity or activity of the IGFBP-3 proteinase. Size exclusion chromatography of bovine aqueous indicates an approximate molecular weight of 260 kDa. Incubation of recombinant IGFBP-3 or serum with partially-purified IGFBP-3ase results in the appearance of low molecular weight fragments of approximately 30 kDa. These fragments are undetectable by western ligand blotting but are readily visualized using an IGFBP-3 specific antibody. Comparison of normal and diabetic vitreous humor reveals the presence of an increased amount of IGFBP-3 proteolytic fragments in the diabetic as compared to control. These findings indicate the presence of a IGFBP-3 proteinase in aqueous and vitreous humors that may be important in regulating ocular homeostasis.

Vitreous and aqueous humors contain a latent proteinase activity that abolishes IGF binding to specific IGF binding proteins.

Several distinct insulin-like growth factor binding proteins (IGFBPs) are present in tissues and fluids of the developing and adult eye. However, the mechanism(s) involved in the regulation of ocular IGFBP levels is unknown. We have now identified an endogenous factor in vitreous and aqueous humors that, when activated by sodium dodecyl sulfate (SDS), abolishes the capacity of specific low molecular weight IGFBPs (i.e. 24-30 kDa) to bind IGF as assessed by western ligand blotting. In contrast, IGF binding to the 46 and 32 kDa IGFBPs (IGFBP-3 and IGFBP-2 respectively) is not affected by the SDS-activated inhibitory factor (IF). Maximal activation of the IF occurs at an SDS concentration of approximately 0.015%. Incubations in the presence of the serine-proteinase inhibitor aprotinin result in marked inhibition of IF activity. Preliminary characterization by ultrafiltration suggests that the IF is large (< 100 kDa) and/or that it is present in a complex. The finding of a factor, most likely a serine proteinase, that specifically abolishes IGF binding to low molecular weight IGFBPs suggests a mechanism for regulating the levels of these IGFBPs and thus the functional activities of IGFs in ocular fluids under normal and/or pathological conditions.