Abstract
Purpose:
The development of age-related macular degeneration (AMD) was previously shown to be related to complement activation. Further, the highly specialized retinal pigment epithelial (RPE) cells play a key role in this disease. The present study addressed the influence of C5a, an anaphylatoxin released by activated complement, on primary porcine (pp) RPE cells.
Methods:
ppRPE cells were isolated from porcine eyes. In order to characterize “older” or “younger” cells in dependence on higher or lower numbers of passages the morphology, the adherence, the activity of β-galactosidase, and the viability were examined. As parameters for changes in the RPE cell metabolism after C5a stimulation the VEGF secretion was measured by ELISA and the presence of reactive oxygen species (ROS) by reaction with nitro blue tetrazolium. The transepithelial electrical resistance (TER) of cells in transwell inserts was recorded by a Volt-Ohm-Meter. Additional immunocytochemistry for terminal complement complex C5b-9 and vitronectin being part and parcel of drusen was performed.
Results:
The histology of the ppRPE cells with ongoing passages revealed a modification from cobblestone to fibroblastic shape associated with loss of melanin granules. ppRPE cells of higher passage numbers had initially a larger proportion of floating to adherent cells. From passage to passage lower VEGF content in the supernatants was measured and TER decreased. A passage-dependant loss of viability and a reduced β-galactosidase activity were detected. By C5a shrunken cell diameters and more dense and also more disordered growth patterns were observed - especially in cells of higher passages. C5b-9 was not detected in response to C5a. Overall staining for vitronectin was low and not altered in response to C5a. The viability was slightly enhanced, the amount of ROS and the VEGF secretion were increased. By C5a higher TER values were recorded.
Conclusions:
Continuous passaging of ppRPE cells was associated with a broad loss of activity. C5a activated the ppRPE cells, resulted in an increased VEGF secretion, and induced oxidative stress that is presumed to be an important risk factor in AMD development. The production of vitronectin and inflammatory markers by C5a was less marked than by general complement stimulation. ppRPE cells seemed to be more susceptible to C5a than ARPE-19 cells and maybe a more suitable model for studying age-related RPE cell responses.
Keywords: 412 age-related macular degeneration •
413 aging •
701 retinal pigment epithelium