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Ping Yang, Muhammad Abu-Rmaileh, Peter Baciu, Glenn J Jaffe; Complement Membrane Attack Complex Release Basic Fibroblast Growth Factor from Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5233. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Complement activation has been increasingly implicated in the pathogenesis of AMD. Complement activation products such as membrane attack complex (MAC) have been detected in Bruch’s membrane (BrM), choriocapillaris intercapillary pillars and on RPE in advanced neovascular and non-neovascular AMD. Basic fibroblast growth factor (bFGF) regulates the activity of vascular endothelial growth factor and is found in choroidal neovascular membranes removed from patients with AMD. Herein, we investigate the effect of complement activation on bFGF release in culture human RPE cells.
Cultured human RPE cells were primed with an anti-RPE antibody (S58) and then treated with 6% C1q-depleted human serum (C1q-Dep) to elicit cell surface MAC formation. Controls included treatment of serum with an anti-C5 antibody (10 µg/ml) to block MAC formation, a heat-inactivated (Hi) C1q-Dep to block complement activation, and C6-depleted human serum (C6-Dep) with or without purified C6 protein (a complement cascade protein downstream of C5a formation) to exclude the possibility that C5a solely accounted for bFGF release. ELISA was conducted on conditioned media. Western blot was conducted on total protein extracts and conditioned media. mRNA expression of bFGF was evaluated by qPCR. Interleukin 6 (IL-6) expression was used as a positive control.
By Western blot, bFGF (18 kDa) in conditioned media was increased in 4.5 hour- and 6 hour-complement-challenged conditioned media, but not in cell lysates when compared with controls that included S58+HiC1q-Dep, and C1q-Dep alone. By ELISA, the bFGF concentration was significantly increased in conditioned media when C6-Dep was reconstituted with C6 but not in C6-Dep alone. Anti C5 antibody significantly attenuated complement-mediated bFGF release. bFGF mRNA levels were not affected, while IL-6 mRNA levels were increased by complement activation.
RPE cell release of bFGF is MAC dependent. Complement-mediated RPE cell release of bFGF may be a co-factor that accounts for neovascularization in AMD. This information will enhance our understanding of the role that complement activation plays to mediate neovascularization, and may elucidate potential therapeutic targets.
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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