Abstract
Purpose :
Subretinal fibrosis affects around one third of patients with neovascular age related macular degeneration (nAMD) and can cause severe vision loss. The underlying mechanism remains poorly defined. We previously reported higher plasma levels of C3a and C5a in nAMD patients with macular fibrosis. The aim of this study was to understand if C3a or C5a can induce Epithelial-Mesenchymal Transition (EMT) in retinal pigment epithelial (RPE) cells.
Methods :
Subretinal fibrosis was induced in mice using a protocol described by Little et al. The presence of C5aR in the fibrotic lesion was examined by immunohistochemistry. Primary murine RPE cells were then treated with recombinant C3a or C5a (10-50 ng/ml) for different durations. Mouse recombinant TGF-β2 (10ng/ml) was used as a positive control for induction of EMT in RPE cells. Expression of epithelial and mesenchymal markers, (vimentin, fibronectin, α-SMA, Slug, and epithelial E-Cadherin) as well as the ERK/AKT and pSMAD2/3 pathways were determined by Western Blot and immunocytochemistry. Collagen contraction and wound healing were used as functional readouts of EMT, and subsequent cytokine and growth factor production and secretion was determine by ELISA and Western Blot
Results :
C5aR was found to be expressed in subretinal fibrotic lesions that were characterized by fibronectin and collagen I positivity. In primary RPE cultures, TGF-β2 treatment (10ng/mL) upregulated the expression of C5aR and increased release of C5a. C5a treatment (96h) increased mesenchymal markers such as fibronectin, α-SMA, vimentin or Slug, and decreased the epithelial marker E-Cadherin. Functional evidence for the role of C5a in EMT was attained when primary RPE treated with C5a displayed increased contractility and migration following treatment.
Interestingly, C5a treatment also increased the production of IL-6 and VEGF, which are known pro-fibrotic mediators. Western Blot revealed that C5a-induced EMT is mediated by Smad2/3 and ERK1/2 signaling pathway activation. C5a-mediated EMT was completely prevented when C5aR was inhibited concomitantly with C5a treatment.
Conclusions :
Our results show that the C5a/C5aR pathway may contribute to subretinal fibrosis through induction of EMT in RPE cells. Targeting this pathway may be a novel approach for the management of macular fibrosis.
This is a 2020 ARVO Annual Meeting abstract.