Abstract
Purpose :
Macular fibrosis and associated vision loss in nAMD patients is well known, however the mechanisms underlying the pathogenesis remain poorly defined in addition to limited treatments available. A potential contributor of fibrosis observed in patients is through epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. The aim of this study was to assess the tyrosine kinase inhibitor (TKI) Nintedanib and its effectiveness in preventing fibrosis in vitro and in vivo.
Methods :
Human retinal pigment epithelial cells (ARPE-19) were used for in vitro studies induced to an EMT phenotype with transforming growth factor beta 2 (TGF-β2), before Nintedanib administration. Various techniques were employed for analysis including morphological assessment, quantitative real-time PCR (qPCR), western blot analysis, and immunofluorescence staining to evaluate the expression of EMT biomarkers. Additionally, a two-stage laser-induced in vivo mouse model of subretinal fibrosis developed in our lab was utilised where the expression of collagen-1 (COL-1), and Isolectin B4 in RPE flat mounts were assessed.
Results :
Stimulation of ARPE-19 with TGF-β2 led to an upregulated expression of several EMT biomarkers including fibronectin, a-smooth muscle actin (α-SMA), and vimentin, while epithelial markers such as ZO-1 were shown to decrease in expression. The metabolic profile, e.g. oxygen consumption rate, of ARPE-19 cells undergoing EMT after stimulation with TGF-β2 was not significantly different to unstimulated cells. Furthermore, ARPE-19 cells undergoing EMT showed increased proliferative capacity and weaker junctional properties between cells. Nintedanib blocked EMT signature, normalised the metabolic profile and proliferative properties of ARPE-19 cells, while gaining epithelial traits such as junctional proteins. In vivo, intravitreal injection of an extended-release formulation of Nintedanib significantly reduced fibrosis and neovascularization in the the two-stage laser-CNV model.
Conclusions :
Nintedanib successfully ameliorates the EMT signature expressed by ARPE-19 cells after stimulation with TGF-β2. Additionally, Nintedanib successfully prevents neovascularization and collagen production at the sight of lesions in a mouse model of choroidal neovascularization. Taken together, the TKI Nintedanib offers a new potential pharmacological treatment option for nAMD patients.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.