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M. Behzadian, E. Duh, J. Yang, A.B. El–Remessy, M. Bartoli, T.F. Miller, R.B. Caldwell; Pigment Epithelium–Derived Factor (PEDF) is Reduced in Retina of Diabetic Mouse; Possible Role of uPAR and MMP–9 Expression . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3815.
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© ARVO (1962-2015); The Authors (2016-present)
To study the mechanism of PEDF regulation in diabetic retinopathy. PEDF is a 50–kDa glycoprotein, expressed at high levels in retinal pigment epithelial cells, but is also found in other retinal cell types including vascular endothelial cells. It is a potent anti–angiogenic and anti–permeability factor, but under hypoxic conditions, PEDF activity is abrogated due to it’s cleavage by matrix metalloproteinases. However, the integrity and the functional role of PEDF in retina has not been fully characterized under diabetic conditions.
Diabetes was induced in 8 weeks old uPAR–/– and their wild type littermates by streptozotocin injection. After 9 weeks, retinal protein extracts were tested by Western blotting for the presence or absence of PEDF. Vitreous samples were examined by zymography for the presence of MMP–2 and MMP–9 and microvascular endothelial cells cultured from bovine retina (BRE cells) were tested for trans–endothelial electrical resistence in the presence of VEGF in combination with various doses of PEDF. Confocal immunofluorescence microscopy was used to evaluate PEDF inhibition of beta–catenin redistribution by VEGF.
Using transgenic uPAR knockout mice we have shown that uPAR is required for diabetes–associated increases in vascular permeability. Vitreous samples examined on gelatin zymograms were all positive for MMP–2, whereas MMP–9 was present only in diabetic wild type but not in diabetic uPAR–/– animals. MMP–9 and pro–uPA are thought to interact on the cell surface to initiate a cascade of proteolytic activities. Western blot analysis showed that PEDF levels were significantly decreased in diabetic wild type compared with non–diabetic mice. In diabetic uPAR–/– mice, PEDF levels remained high, indicating that uPAR plays a major role in decreasing PEDF levels under the diabetic condition, probably through activation of MMP–9. PEDF at low concentration blocked VEGF–induced paracellular permeability (measured by TER fall) in BRE cell monolayers. At high concentrations, the PEDF effect was reversed, enhancing the VEGF–induced permeability. Finally, confocal microscopy showed that PEDF blocks VEGF–induced redistribution of beta–catenin in BRE cells.
We have shown that diabetes–induced increases in retinal permeability and MMP–9 expression accompany decreases in PEDF levels in the diabetic retina. The process is abrogated in the absence of functional uPAR, indicating the involvement of uPA/uPAR system in PEDF activity.
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