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G Abboud, S He, SJ Ryan, DR Hinton; Biphasic Modulation of RPE Migration by Thrombospondin-1 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4518.
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Purpose: Thrombospondin-1 is a multifunctional matricellular glycoprotein that is expressed by a variety of cell types, including RPE, and that has been shown to modulate cellular migration. The present study aims to determine how RPE cytoskeletal morphology and cellular migration are affected when TSP-1 levels are altered by either overexpression or exogenous administration. Methods: Two replication-deficient adenoviral vectors (Ad) were generated: the first, Ad-GFP, contains a cytomegalovirus (CMV) promoter-driven green fluorescent protein (GFP); the second, Ad-GFP/TSP1, is an Ad-GFP vector that contains a full-length human TSP-1 cDNA driven by a second CMV promoter. Human fetal RPE (HFRPE) cells were transfected either with Ad-GFP (control) or with Ad-GFP/TSP1. TSP-1 overexpression was ascertained by Western blotting using TSP-1-specific polyclonal antibody. Other HFRPE cells were exposed to increasing concentrations of platelet-derived human TSP-1 (0.1, 1.0, 3.0, 5.0, 7.0, 25.0, 50.0nM). Changes in cytoskeletal morphology were assessed by observing the cellular distribution of F-actin and cytokeratin under confocal microscopy, using phalloidin staining and cytokeratin-specific polyclonal antibody respectively. Cell migration was determined by means of the Boyden chamber assay with and without the addition of 10ng/ml recombinant basic fibroblast growth factor (bFGF) as cellular motogen. Results: Adenovirus-mediated TSP-1 overexpression increased RPE migratory ability by 50% (uncoated wells) and 67% (wells coated with fibronectin) but did not affect the cellular distribution of F-actin and cytokeratin. When tested alone, exogenous TSP-1 inhibited RPE migration at concentrations of 7nM or less (maximal inhibition of 77% at 3nM) but stimulated migration at elevated concentrations (25, 50nM). Exogenous TSP-1 at concentrations of 7nM or less was also effective at inhibiting bFGF-induced migration. Conclusion: TSP-1 is an effective inhibitor of RPE cellular migration at doses in the lower nM range, but it becomes stimulatory when it is overexpressed and when its concentration rises above 25nM. Since RPE cells are a natural source of TSP-1 and since RPE cells possess receptors for this glycoprotein, we postulate that RPE migration could potentially be regulated by an autocrine loop involving TSP-1, whereby increasing production of TSP-1 by dedifferentiated RPE would induce their proper migration. TSP-1 should be further evaluated as a potential target of therapy for proliferative ocular disease.
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