Isolation and characterization of vitreous insulin-like growth factor binding proteins.

Proliferative diabetic retinopathy (PDR) is a late-stage complication of diabetes in which fibrovascular tissues emerge from the retina and exert tractional forces that can cause retinal detachment.1 Though complex and incompletely understood, PDR is ultimately a cellular disorder involving a progression of specific activities that minimally include cell translocation from the retina, cell division, and the generation of tractional forces.2 There is considerable interest in identifying the causal cell types and the stimuli leading to their pathogenic activities because the ability to arrest or attenuate any of these processes would represent a significant gain in control of this complication. Immunohistochemical studies of diabetic epiretinal tissues have identified a number of different cell types, including glia, immune cells, retinal pigment epithelial cells, and fibroblastlike cells of unknown origin.3–8 Muller cells, the principal retinal glia, are consistently identified in diabetic fibrovascular scar tissues using the traditional immunocytochemical markers glial fibrillary acidic protein and glutamine synthetase.3,6,8,9 In addition, more recent studies from this laboratory revealed that the fibroblastlike cells mentioned are also derived, at least in part, from phenotypically altered Muller cells.9 Described was a progressive loss of glial-specific proteins and de novo expression of alpha smooth muscle actin ending with myofibroblastlike cell types that are unrecognizable as glia in a process that closely resembles phenotype changes previously described in primary cultures of Muller cells.10,11 There is also abundant circumstantial evidence indicating that Muller cells are a source of the tractional forces that cause retinal detachment in PDR. Although freshly isolated, phenotypically recognizable Muller cells lack the capacity to generate tractional forces, this is not the case with the fibroblastlike phenotype described.11,12 In concert with a phenotype change, and in particular with de novo expression of α-smooth muscle actin, Muller cells acquire the capacity for tractional force generation in culture. Systematic study of Muller cell tractional force generation in vitro revealed that this activity is not constitutive but is instead stimulated by certain exogenous promoters, including ligands, in the insulin-like growth factor and platelet-derived growth factor systems.11,12 Consistent with these findings, tractional force–stimulating activity to which Muller cells respond is present in diabetic, but not in normal, vitreous, and studies with growth factor–neutralizing antibodies attributed most of this activity to IGF-related ligands.13 As a consequence of these findings, there is now considerable interest in gaining an improved understanding of vitreous changes in diabetes and, in particular, those that result in increased IGF biological activity. Several laboratories have examined diabetes-associated changes in vitreous IGF-I and IGF-II levels, reporting increases varying between 150% and 300%.14–20 However, the values reported for normal vitreous correlate poorly with the observed absence of biological activity, leading us to speculate that a control mechanism attenuates vitreous ligands.13,21 The insulin-like growth factor system also contains six high-affinity insulin-like growth factor binding proteins (IGFBPs) that can potentiate or inhibit IGF activities and can also have direct growth factor–independent effects.22–27 Several laboratories used ELISA-type assays to examine IGFBP levels in normal and diabetic vitreous and reported detecting IGFBP-2 and IGFBP-3.15,18,20 We have since examined the effects of all six IGFBPs on Muller cells generating tractional forces in response to IGF and determined that IGFBP-2 and IGFBP-3 are potent inhibitors of IGF-I and IGF-II activities.28 Together these observations led us to propose that IGFBPs in normal vitreous function as a growth factor sink, limiting the concentration of free ligand and thus controlling IGF biological activity.28 However, there are several confounding elements to this hypothesis, including a report suggesting that IGFBP-3 in normal vitreous is cleaved to an inactive form by local proteases.29 In addition, IGFBP levels in vitreous reportedly increase rather than decrease in diabetes, suggesting that these changes may be pathogenic rather than protective.15,18,20 The goal of this project was to determine which IGFBPs are present in normal vitreous and to determine which of these retain the capacity to bind IGF ligands and thus modulate growth factor biological activity.