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Carolyn Trepp, Esther Melo Herráiz, Roberto Iacone, Volker Enzmann; Effect of the serine protease HtrA1 overexpression on RPE phagocytosis in an in vitro disease model. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2287.
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© ARVO (1962-2015); The Authors (2016-present)
Recent studies have shown a strong association between the serine protease HtrA1 and age-related macular degeneration (AMD). Increased HtrA1 expression in retinal pigment epithelium (RPE) has been shown to cause extracellular matrix degradation and destabilization of the Bruch’s membrane. However, little is known about functional effects of HtrA1 overexpression on RPE. To investigate these effects we established an in vitro model of polarized RPE overexpressing HtrA1 where phagocytic activity, a key function of RPE, could be assessed.
Human fetal RPE cells cultured on laminin-coated transwell membranes were infected either with the wild type HtrA1- or the HtrA1 Serine-Alanine mutation (S328A)-GFP IRES construct two weeks after seeding. After five weeks of growth in vitro phagocytosis assay was performed. Thereby, cells were incubated with CY5-labelled photoreceptor outer segments (POS) isolated from porcine retinas for six hours. POS internalization was observed by live cell imaging and uptake was quantified by confocal microscopy. Cells containing at least three particles where considered to actively phagocyte POS.
Transfection efficiencies of the constructs were assessed by quantification of GFP positive cells. In both conditions transfection efficiencies reached at least 70%. Live cell imaging revealed a polarized phenotype of S328A cells with longer and more abundant microvilli compared to HtrA1 cells. In the HtrA1 condition POS uptake concentrated on the non-transfected cells. Furthermore, the HtrA1 transfected cells showed a significantly reduced phagocytic activity reaching values of 9.7 % ± 6.8% compared to 21.2 % ± 11.4 in S328A cells (mean % of activity compared to control cells, p=0.0002).
The established model has proven to be a reliable tool to assess phagocytic activity in RPE cells overexpressing HtrA1. Our results confirm the hypothesis that diseased RPE have an impaired phagocytic activity and this is directly associated to the apical membrane condition. This model could be employed in the functional readout of possible therapeutic modalities targeting HtrA1.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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