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C. Guidry, J.L. King; Insulin-Like Growth Factor Binding Protein Biosynthesis by Normal, Reactive and Fibrocontractive Müller Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2966.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Müller cells, the principal glia of the retina, are involved in fibroproliferative ocular diseases such as proliferative diabetic retinopathy and are capable of generating tractional forces in response to insulin-like growth factors I or II. Previous studies revealed that the normal complement of vitreous IGFBPs is altered in diabetes and that several of these proteins have the capacity to modulate IGF-I and IGF-II activities. The present study sought to confirm that normal porcine retina is a biosynthetic source of IGFBPs and test the hypotheses that 1) IGFBP biosynthesis by Müller cells is modulated with cell phenotype and 2) Müller cells are a source of IGFBP-modulating proteases. Methods: Extracellular matrix contraction was examined using an established tissue culture model involving incubation of IGF-I-stimulated porcine Müller cells on gels formed with type I collagen. RT-PCR and Northern blots were performed on total RNA preparations from normal porcine retina, reactive Müller cells (culture passage 1) and fibroblastic Müller cells (culture passage 6). Experiments to detect IGFBP degradation involved incubating recombinant IGFBPs 1 through 6 with cultures of fibroblastic Müller cells with analysis by Western ligand blotting. Results: Insulin-like growth factor analogs (R3IGF-I, R6IGF-II) that lack affinity for IGFBPs were consistently more potent than the native ligands (IGF-I, IGF-II) in stimulating extracellular matrix contraction, suggesting that Müller cells produce IGFBPs. RT-PCR analyses with IGFBP-specific primers and RNA preparations from normal porcine retina revealed the presence of message for IGFBPs 3, 5 & 6 and the absence of message for IGFBPs 1, 2 & 4. Both reactive and fibroblastic Müller cells were positive for IGFBPs 3, 4, 5 & 6, but negative for IGFBPs 1 & 2. Northern blots to assess relative abundance of these mRNA revealed that expression of IGFBPs 3, 4, 5 and 6 is essentially unchanged between normal retina and reactive Müller cells, but there is significant upregulation of IGFBP 3, 4, and 6 expression in fibroblastic Müller cells. Finally, IGFBP coincubation with fibroblastic Müller cells in culture did not result in substantial protein degradation as assessed by Western ligand blots. Conclusions: Based on the abundance of IGFBP-specific mRNA, Müller cell biosynthetic potential in the early, reactive phenotype is unchanged from that of the normal retina. However, Müller cells of the fibroblastic phenotype upregulate expression of at least three IGFBP species, but do not produce IGFBP-modulating proteases.
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