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Paul Kay, Yit C. Yang, Luminita Paraoan; Polarized secretion of Cystatin C from human fetal retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1604.
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© ARVO (1962-2015); The Authors (2016-present)
Cystatin C is a cysteine protease inhibitor expressed by the retinal pigment epithelium (RPE). The previously characterised variant B version of this protein has been associated with an increased risk of developing exudative age-related macular degeneration (AMD). The aim of this study was to optimize the culture and manipulation of human fetal RPE (hfRPE) monolayers, in order to analyze the secretion of the endogenous cystatin C, as well as eGFP fused wild-type and variant B cystatin C constructs.
hfRPE cells obtained from donors at 16 to 18 weeks of gestation (kindly donated by S. Miller, NEI) were seeded onto extracellular matrix coated 1.12cm2 transwell inserts and maintained in 5 % FCS containing media until monolayers consisting of a hexagonal array of densely packed, pigmented cells were formed. Fusion constructs containing the wild-type or variant B ORF fused to eGFP were transfected into hfRPE cells using Fugene 6 at a 3:1 ratio. Transepithelial resistance (TER) measurements across the monolayer were measured using an epithelial volt-ohmeter. The protein content of conditioned media collected from the apical and basal facing chambers was analysed by Western blot. Average protein expression was determined by densitometry, and differences between groups were analyzed by paired T-test.
30 days after seeding, TER measurements across hfRPE cell sheets were >200Ωcm2, indicating the presence of an impermeable, highly polarized monolayer. Endogenous cystatin C was present in media harvested from both sides of the monolayer, and was more abundant in the basal chamber, suggesting preferential basolateral secretion. Transfections of the hfRPE cells were successful, resulting in expression of the exogenous eGFP tagged versions of both wild-type and variant B cystatin C. The secretion pattern of these fusion proteins was similar to that of the endogenous protein. Pigment epithelium derived factor (PEDF) was secreted from monolayers preferentially into the apical compartment, as previously reported.
Our results validate the use of hfRPE cells to create monolayers that closely resemble the physiological function of the native RPE, in relation to secretion of cystatin C. We have demonstrated that these cells can be transfected to express exogenous cDNA constructs, and that cystatin C is secreted in a polarized fashion. These findings support the hypothesis of an extracellular function for cystatin C, likely involving regulation of proteolytic activity and maintaining the structure and function of the Bruch’s membrane/choroid. Impaired secretion of cystatin C due to mutation (variant B), or age could therefore contribute to the development of AMD.
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