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Angela Armento, Sabina Honisch, Anke Jacob, Dragana Trifunovic, Marius Ueffing; Complement factor H (CFH) loss leads to a local complement system activation and impaired cell balance in the RPE cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1242. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Age related macular degeneration (AMD) is the leading cause of blindness in the elderly population. Although the etiology of AMD is still not fully elucidated, complement system (CS) dysregulation is involved and genetic variants of complement genes, like CFH, are major drivers of AMD. The contribution of CFH, the key regulator of the CS, to local complement activation in the retina and its influence on RPE cells homeostasis still remain vague. We hypothesized that CFH plays a protective role in the RPE and its absence contributes to AMD pathology.
To investigate the role of CFH, we used shRNA to silence CFH in hTERT-RPE-1 cells and evaluated by RT-qPCR (n=3, FC ± SEM) whether CFH reduction has an impact on the expression of other complement factors and inflammatory cytokines. We also used ELISA, to establish if CFH reduction influences CS activation. The effects of CFH depletion on RPE cells were assessed, under normal condition and under H2O2 -induced oxidative stress, via cell viability, cell density, cytotoxicity and caspase activity assays.
CFH depletion led to a significant upregulation of several genes involved in the activation of the CS including complement component 3 (C3; 6.3 ± 0.9, p=0.005) and complement factor B (CFB; 12.9 ± 4.2, p=0.05). At the same time, the expression of pro-inflammatory cytokines like C-C Motif Chemokine Ligand 2 (CCL2) was increased as well as the pro-angiogenic vascular endothelial growth factor (VEGF). Reduced level of CFH translated in a significant increase in C3b concentration (13.6 ± 2.2 ng/ml, p=0.04) compared to controls (7.2 ± 0.8 ng/ml), indicating a local complement activation mediated solely by RPE cells. Moreover, CFH downregulation severely affected RPE cells homeostasis, leading to increased cytotoxicity in both, normal and H2O2- stressed RPE cells. These effects were accompanied by a higher caspase 3/7 activity (2.9 ± 0.1, p=0.001). Interestingly, cell viability was affected only after H2O2 treatment in absence of CFH.
Our data support the hypothesis that CFH is protective in RPE cells. As a result of CFH depletion, RPE cell balance is strongly impaired due to the local activation of the complement system and a pro-inflammatory environment. These findings may help elucidating the function of CFH in the retina and the role of complement in the pathogenesis of AMD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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