Abstract
Purpose :
Increased expression of pro-inflammatory cytokine TNFα is a hallmark of several angiogenic retinal diseases including proliferative diabetic retinopathy (PDR) and wet age-related macular degeneration (AMD). Previous research has shown the effect of high glucose on RUNX1 expression, identifying RUNX1 as a mediator of aberrant retinal angiogenesis. We have identified TNFα-induced up-regulation of Runt-related transcription factor 1 (RUNX1) in human microvascular retinal endothelial cells (HMRECs). We have defined the primary components of the TNFα-JNK-RUNX1 pathway and have demonstrated that small molecule inhibitors can target this pathway, indicating that it is a novel, targetable pathway of potential therapeutic interest.
Methods :
HMRECs were treated in supplemental growth factor free media for 12 and 72 hours with various treatments, including D-glucose (30 mM), TNFα (5 ng/ml), and commercially available small-molecule JNK activators (anisomycin) and inhibitors of TNFα (CAY10500), JNK (Withaferin A, SP600125, TCS JNK 6o), AP-1 (SR11302) and RUNX1 (Ro5-3335). Effects were measured by qRT-PCR and Western blot analyses, with antibodies against RUNX1 JNK, p-JNK (all Santa Cruz Biotechnology), and β-actin (Cell Signaling Technology) as housekeeping control. One-way ANOVA was used for statistical analysis.
Results :
TNFα significantly increased RUNX1 RNA (2.4 ± 0.18 fold, p < 0.0005, n = 6, 48h) and protein expression (2.0 ± 0.36 fold, p < 0.005, n = 2, 72h) in HMRECs. Direct activation of JNK using anisomycin caused a significant increase in the mRNA levels of JNK (2.53 ± 0.25, p < 0.05) and RUNX1 (3.16 ± 0.44 fold, p < 0.0005) over 48 hours. Co-treatment of HMRECs with inhibitors of TNFα and JNK caused a significant decrease in RUNX1 mRNA and protein levels compared to TNFα alone. Similarly, co-treatment ofAP1 inhibitor and TNFα resulted in a significant decrease in RUNX1 expression (1.55 ± 0.39, p < 0.05, n =6) at 48 hours (non-significant compared to controls).
Conclusions :
These findings demonstrate the existence of a novel pathway in HMRECs that is activated by TNFα, modulated by JNK and results in increased RUNX1 expression. RUNX1 has been identified as a significant mediator of aberrant retinal angiogenesis. By identifying the molecular mediators of TNFα-induced regulation of RUNX1 expression, we have shown specific therapeutic targets that may regulate pathologic angiogenesis.
This is a 2020 ARVO Annual Meeting abstract.