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O. Kazanskaya, T. Möller, G. Thumann; Recombinant PEDF Proteins Produced in Eucaryotic Cells: Potential Use for Anti-Angiogenic Therapy of AMD. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4754.
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Pigment epithelial-derived factor (PEDF) is a potent multifunctional factor that inhibits angiogenesis and acts as a neuroprotective and maintenance factor for the neural retina. Since AMD includes angiogenesis and retinal cell degeneration, PEDF would be an ideal candidate for AMD gene therapy. We have previously shown that using non-viral protocols pigment epithelial cells can be efficiently and stably transfected with PEDF constructs and that the secreted PEDF is biologically active as determined by the up-regulation of the zinc receptor gene expression. Here we have examined the biological activity of recombinant PEDF on vascular endothelial cell migration and sprout formation.
6-His tagged PEDF expressed in hRPE was purified by Ni-NTA affinity chromatography. The purified rPEDF was added to cultures of human umbilical vein endothelial cells (HUVEC) for the determination of sprouting. Cell migration was determined by enumerating the number of cells that migrated to the underside of Transwell inserts in 10 high magnification fields. Viability was determined by enumerating dead cells in endothelial cell cultured in the presence of low (0.2%) serum.
Recombinant PEDF reduced cumulative sprout length per spheroid in the presence of VEGF from 2.80±0.15 mm to 1.35±0.14 mm and decreased significantly migration of HUVECs through Transwell inserts in presence of VEGF, the number of cells per field was reduced from 215±39 to 97±21. In addition, rPEDF increased the percent of dead cells in HUVEC cultures from 14±4 to 48±3 when cultured in the presence of 0.2% serum and 2ng/ml VEGF.
The results presented here show that rPEDF produced by transfected RPE cells has the same biological activity as commercially available PEDF. Transplantation of cells transfected with the PEDF gene should restore a permissive subretinal environment for RPE and photoreceptor maintenance while inhibiting blood vessel growth.
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