Abstract
Purpose :
Our previous work revealed dysregulated autophagy in aging and in glaucomatous TM cells. The purpose of this study was to gain more insight in the role of autophagy in the TM pathophysiology
Methods :
Whole-transcriptome expression profiling was performed in three primary cultures of human TM transfected with siRNA targeting the autophagy genes Atg5 and Atg7, using Affymetrix Clariom D arrays. Cells transfected with scrambled siRNA were used as control. Gene and functional analyses were conducted using Partek Flow and Metacore software. Gene array data was confirmed by qPCR, ELISA and/or WB in additional strains with genetic (siLC3, siAt5, siAtg7) or pharmacological (3-MA, BafA1) inhibition of autophagy. Treatment of TM cells with TGFβ1 and TGFβ2 was performed for 48 hours at increasing concentrations (0-10 ng/mL). Autophagy was evaluated by WB, live imaging (GFP-LC3), and by electron microcopy. siRNA targeting Smad2/3 was used to abolish TGFβ-signaling
Results :
Microarrays and qPCR showed the upregulated expression of TGFβ2 (2.04 fold, p=0.02; 2.11±0.88 fold, p<0.05, n=3) and BAMBI (2.17 fold, p=0.01; 2.14±0.85 fold, p<0.05, n=3) in siAtg5/7-transfected hTM cells compared to siNC. Increased TGFβ2 in autophagy-deficient cells was confirmed by ELISA (0.16±0.05 vs 0.12±0.01 pg/μL/μg, p<0.05, n=5). Interestingly, genetic and pharmacological inhibition of autophagy significantly reduced (p<0.05, n=3) the constitutive and TGFβ-induced mRNA and protein levels of the fibrotic markers α-SMA, fibronectin and Col I. In agreement, functional analysis identified regulation of TGFβ-mediated-endothelial to mesenchymal transition as the top cellular pathways affected in autophagy-deficient TM cells. At the same time, TGFβ treatment dysregulated autophagy in TM cells in a dose-dependent manner, which was characterized by higher presence of GFP-LC3 puncta and autophagic vacuoles, elevated LC3-II levels (TGFβ1: 139±19; TGFβ2: 158±32, p<0.05, n=4) and reduced p62 (TGFβ1: 59±8; TGFβ2: 63±9, p<0.05, n=4). Silencing Smad2/3 expression completely blocked the TGFβ-induced increase in LC3-II
Conclusions :
Our data shows, for the first time, an intricate interplay between autophagy and TGFβ signaling, and a role of autophagy in regulating fibrogenesis in TM cells. The implication of autophagy in the induction of the fibrotic response opens a novel area for investigation of therapeutic targets for amelioration of fibrosis in glaucoma
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.