Abstract
Purpose :
Diabetic retinopathy is multifactorial with many precursors. Iron, Interleukin (IL)-17A, and oxidative stress have been previously reported to play a role in diabetic retinopathy. Six-transmembrane epithelial antigen of prostate 4 (STEAP4) is a ferrioxidase that can be induced by IL-17A or iron to induce oxidative stress or angiogenesis. Yet the role of STEAP4 in diabetic retinopathy is unknown. We hypothesized that STEAP4 plays a role in retinal oxidative stress and vasculogenesis, which are precursors to diabetic retinopathy.
Methods :
Retinas of non-diabetic and streptozotocin (STZ)-induced diabetic C57BL/6 mice were collected 2-months post-diabetes. STEAP4 expression was analyzed by qPCR, Western immunoblot, and immunofluorescent staining (n=5/group). Next, retinas of non-diabetic and STZ-diabetic mice (n=5/group) was stained with 4HNE and microscopically analyzed to quantify oxidative stress. Additionally, untreated or STEAP4 inhibited human Muller glia or photoreceptor-like cone cells (661W) were stimulated with recombinant IL-17A (10ng/ml) for 16h for H2DCFDA analysis of oxidative stress. Finally, untreated or STEAP4 inhibited human retinal endothelial cells (hREC) were incubated with recombinant IL-17A (10ng/ml) and VEGFA (100ng/ml) for 16h, for protein analysis of angiogenesis. Statistical analysis was performed using a 2-way ANOVA and then an unpaired student’s t-test.
Results :
STEAP4 mRNA was increased by 2-fold in the retinas of STZ-diabetic mice. Constitutive STEAP4 protein in the retina was significantly increased in STZ-diabetic mice (p<0.05). Per immunofluorescent staining, areas near photoreceptor cells were both STEAP4+ and 4HNE+ (a fluorescent indicator of oxidative stress) in the retinas of STZ-diabetic mice. Reactive oxygen species production was induced in both human Muller glia and 661W cells when stimulated with IL-17A, which was significantly decreased when STEAP4 was inhibited. Finally, IL-17A+VEGFA induced angiogenesis in hREC, which was significantly decreased when STEAP4 was inhibited.
Conclusions :
These results support our hypothesis that STEAP4 plays a role in retinal oxidative stress and vasculogenesis. Since these are precursors to diabetic retinopathy, further long-term in vivo studies will be needed to define the role of STEAP4 in diabetic retinopathy.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.