Abstract
Purpose :
Age-related macular degeneration (AMD) is the major cause of impaired vision in developed countries. The first indication of this disease is the formation of basal deposits (BD) between the retinal pigment epithelium (RPE) and the Bruch’s membrane. We recently demonstrated the roles of complement C3a and extracellular matrix (ECM) turnover in an in vitro model for deposit formation in inherited macular degeneration using primary mouse RPE cells. For this study we tested the hypothesis that C3a can cause the formation of BD in vitro by human RPE cells.
Methods :
Human fetal RPE cells were isolated from the eyes of 14-18 week old fetuses and grown on transwells for 2 weeks when homogeneous pigmentation was observed. RPE cultures were treated with different doses of recombinant human C3a protein every 72 hours for 1, 2 or 4 weeks. The formation of BD was characterized by transmission electron microscopy and immunofluorescence. The presence of complement components, ECM proteins and other proinflammatory molecules was quantified by inmunofluorescence and ELISA. Matrix metalloproteinase (MMP) activity was measured by zymography.
Results :
Electron micrographs showed that RPE treated with C3a in vitro accumulate collagen fibers and wide-spaced collagen under the basal membrane. Immunostaining of the cultures demonstrated that fibronectin, collagen IV and collagen VI, the main components of basal laminar deposits in AMD patients, accumulate under the RPE cells treated with C3a. IL-6 was elevated while the MMP-2 activity was diminished in a dose-dependent manner in media from the C3a treated RPE cells.
Conclusions :
Human fetal RPE cells produce BD in response to stimulation with C3a, further suggesting a key role for C3a in the formation of sub-RPE deposits.
The accumulation of ECM proteins under the RPE plus the diminished MMP-2 activity in culture media demonstrates that C3a can regulate the ECM formation and turnover by the RPE in vitro. This model provides a valuable tool to study the pathogenesis of the RPE in early stages of AMD as well as the role of the complement system in the formation of BD. Also, it may be a useful system to test complement-modulating drugs to prevent BD formation in macular degenerations.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.