Long-term Activation of NADPH oxidase 4 Leads to Endothelial Injury via Mitochondrial Impairment in Diabetic Retinopathy

Abstract

Purpose : Previously we reported that NADPH oxidase 4 (Nox4) is upregulated in retinal endothelial cells during diabetes and contributes to vascular leakage and acellular capillary formation. However, the mechanisms by which Nox4 upregulation is implicated in vascular pathology remain unclear. Herein, we investigated the impact of long-term upregulation of Nox4 on mitochondrial activity, redox status, cellular senescence and apoptosis in vascular endothelial cells in vivo.

Methods : Nox4^EC-Tg mice were generated by crossing lox-Stop-lox-human Nox4 Tg mice with Tie2-cre mice. Diabetes was induced with streptozotocin in Nox4^EC-KO and WT mice. Primary microvascular ECs (BMECs) were isolated from mouse brain. Cellular senescence and apoptosis were evaluated by β-Galactosidase staining using Senescence Detection Kit and TUNEL assay, respectively. Mitochondrial respiratory function was measured with Cell Mito Stress Test Kit by Seahorse XFe24 Analyzer. Mitochondrial potential (ΔΨm) was evaluated by fluorescent cationic dyes JC-1 and TMRE. Mitochondrial superoxide production, glutathione (GSH) level, and lipid peroxidation were examined.

Results : Nox4^EC-Tg mice developed tortuous retinal blood vessels, focal vascular leakage, acellular capillary formation, and retinal degeneration. Compared to WT controls, BMECs isolated from Nox4^EC-Tg mice demonstrated enhanced apoptosis and increased number of senescent cells. This was accompanied by elevated MitoSOX level, reduced GSH levels, and increased lipid peroxidation. Furthermore, ΔΨm measured by JC-1 and TMRE showed a 50% reduction, and mitochondrial respiratory function, measured by oxygen consumption rate (OCR), was significantly decreased in Nox4 overexpressing BMECs compared to WT cells. In addition, BMECs isolated from diabetic WT mice also demonstrated enhanced apoptosis, elevated MitoSOX and lipid peroxidation levels, and reduced ΔΨm, to an extent comparable to that observed in Nox4-overexpressing cells. There changes were largely prevented or significantly reduced in Nox4-deficient BMECs from diabetic Nox4^EC-KO mice.

Conclusions : These results suggest that long-term upregulation of Nox4 in endothelial cells impairs mitochondrial function and disrupts mitochondrial integrity resulting in enhanced oxidative stress, cellular senescence and apoptosis, contributing to vascular pathology in diabetic retinopathy.

This is a 2020 ARVO Annual Meeting abstract.