Vitreous IGFBP-3 effects on Müller cell proliferation and tractional force generation.

Proliferative diabetic retinopathy (PDR) is a late-stage complication of diabetes in which fibrovascular tissues emerging from the retina exert tractional forces that cause retinal detachment.1,2 PDR is ultimately a cellular disorder with proliferation and tractional force generation as major pathogenic activities.2 There has been considerable interest in identifying the causal cells and the stimuli driving these pathogenic activities as the ability to arrest them would represent a significant gain toward controlling this complication. Immunohistochemical studies of diabetic epiretinal tissues identified different cell types including glia, immune cells, retinal pigmented epithelial cells, and fibroblast-like cells of uncertain origins.3–8 Muller cells, the principal retinal glia, are consistently identified in diabetic fibrovascular scar3,6,8,9 and there is abundant evidence to indicate that they are a source of the fibroblast-like cells in PDR.9–11 Systematic studies of Muller cell tractional force generation in vitro revealed that this activity develops in concert with acquisition of the fibroblast-like phenotype and is not constitutive, but is stimulated by certain exogenous promoters including members of the insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) families.10,11 In addition, IGFs and PDGF are reported to be Muller cell mitogens and may play important roles in driving cell proliferation.12–14 Recent studies from this laboratory examined vitreous from normal and diabetic eyes for the ability to stimulate Muller cell tractional force generation in vitro.15 It was determined that normal vitreous induces little or no response while samples from patients with diabetes or PDR stimulate significant responses, the magnitude of which correlate with disease severity. More recently, we detected similar vitreous changes in swine with chemically induced diabetes suggesting that the vitreous changes may precede the development of retinal disease.16 In addition, studies with growth factor-neutralizing antibodies were able to attribute the stimulatory activity in human diabetes to IGFs rather than PDGF. 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. Interestingly, when one considers the IGF concentrations reported for normal vitreous,17–22 there should be higher levels of biological activity than we observe15,23 suggesting that the growth factors are in some way controlled or attenuated. This led us to speculate that diabetes-associated increases in vitreous IGF activity can arise from increases in growth factor levels or from loss of normal control. We have since determined that the insulin-like growth factor binding proteins (IGFBP) present in normal vitreous, IGFBP-2 and IGFBP-3, are able to neutralize IGF-I and IGF-II effects on Muller cell tractional force generation.24 Our studies have provided a better understanding of IGFBP-2 effects on Muller cells, but IGFBP-3 is complicated by the fact that it exists in vitreous as a proteolytic fragment of the intact protein.25 The fragment appears to originate in plasma, cross the blood-vitreous barrier, possesses growth factor affinity and thus has the potential to influence IGF biological activity. However, our understanding of its effects on Muller cells is otherwise limited by the absence of experimental evidence. The goal of this study was to evaluate the direct and indirect effects of the vitreous IGFBP-3 fragment on Muller cell proliferation and tractional force generation to improve our understanding of its role in vitreous biochemistry in normal and disease states.