Abstract
Purpose :
Pro-inflammatory cytokine TNFα is increased in several angiogenic retinal diseases, including proliferative diabetic retinopathy (PDR). Our research on human micro-vascular retinal endothelial cells (HMRECs) has shown that high glucose and TNFα stimulation cause an additive effect on Runt-related transcription factor 1 (RUNX1) expression. This study aims to determine the role of RUNX1 in the modulation of the TNFα-stimulated oxidative stress response.
Methods :
HMRECs were treated in supplemental growth factor free media for between 1 and 72 hours with D-glucose (30 mM), TNFα (5 ng/ml), RUNX1 inhibitors and combinations of these. Effects on gene expression were assessed by qRT-PCR. The Seahorse XF24 Mito Stress Test was used to determine the effects of these treatment conditions on mitochondrial respiration. One-way ANOVA was used for statistical analysis.
Results :
TNFα+glucose significantly reduced both the spare (p< 0.0001) and maximal (p< 0.01) respiration capacity while increasing oxygen consumption (p<0.05) in the Mito Stress Test. Additionally, after 48 hours TNFα significantly increased the expression of TNF-R1 (1.3 ±0.1 fold, p<0.05), while co-treatment of TNFα and RUNX1 inhibitor brought RUNX1 and TNF-R1 to basal levels (1.2 ±0.2 fold, ns; and 0.9 ±0.1 fold, ns; respectively). Superoxide dismutase 2 (SOD2) mRNA expression is significantly increased by TNFα+glucose stimulation (6.1 ±0.1 fold, p<0.0001, 48h). SOD2 expression is also increased by TNFα alone over a 1 - 72h timecourse analysis (5.7 ±0.2 fold, p<0.0001, 48h) and significantly reduced by RUNX1 inhibition (0.79 ±0.1 fold, p<0.05, 48h). In contrast, BCL-2 mRNA is upregulated with RUNX1 inhibition (1.4 ±0.2 fold, p<0.005, 48h).
Conclusions :
These results suggest that while TNFα and glucose reduce mitochondrial respiratory capacity, inducing oxidative stress, SOD2 is significantly upregulated by TNFα-RUNX1 signaling and downregulated via RUNX1 inhibition. This indicates that SOD2 is acting as a protective factor to remove toxic superoxide in the presence of pro-inflammatory TNFα. Moreover, further pro-survival compensatory mechanisms appear to counter the decrease in SOD2 expression due to RUNX1 inhibition, via upregulation of anti-apoptotic BCL-2. This results in greater cell survival capability, even in a high oxidative stress environment.
This is a 2021 ARVO Annual Meeting abstract.