Overexpression of NADPH Oxidase 4 Induces Endothelial Senescence by Impairing Mitochondrial Biogenesis and ATF4 Activation

Abstract

Purpose : NADPH oxidase 4 (Nox4) is the major source of oxidative stress in endothelial cells (ECs) and is upregulated in diabetic retinopathy (DR). Increased cellular senescence has been observed in retinal blood vessels and contributes to DR pathogenesis. In this study, we investigated the role and mechanism of Nox4 in inducing retinal endothelial and vascular senescence in DR.

Methods : Primary brain microvascular endothelial cells (BMECs) and retinal microvessels (RMVs) were isolated from wild-type (WT) and humanized EC-specific Nox4 transgenic (hNox4^EC-Tg, referred to as TG) mice. Cellular senescence was evaluated by (1) senescence-associate β-galactosidase activity (SA-β-GAL); (2) expression of senescence-related genes quantified by quantitative real-time RT-PCR (qPCR); (3) senescence-associated secretory phenotype by measuring the secretion of IL-1β and hydrogen peroxide (H₂O₂) by ELISA or a H₂O₂ Assay Kit, respectively.

Results : TG-BMECs demonstrated a significantly increased number of SA-β-GAL positive cells compared with WT-BMECs. Consistently, expression of senescence markers including p21, Cdk6, Cdkn2a (p16) increased drastically in BMECs, and in RMVs, isolated from TG mice. Moreover, the levels of IL-1β and H₂O₂ secreted from TG-BMECs were significantly higher than that from WT-BMECs. To determine the mechanism of Nox4-mediated cellular senescence in ECs and retinal vessels, we examined mitochondrial biogenesis and the activating transcription factor 4 (ATF4)/p16 pathway, which have been linked to premature senescence in ECs and diabetic microvascular complications. We found that the expression of genes involved in mitochondrial biogenesis, including Opa1, Mfn2, Nrf1, Ppargc1β, and Tfam, was substantially decreased by 40-60% in TG-BMECs. A significant reduction in Mfn2 expression was also observed in RMVs from TG mice. Furthermore, overexpression of Nox4 led to an increased activation of the ATF4/p16 pathway. Inhibition of H₂O₂ by catalase or reducing oxidative damage by a potent radical-trapping antioxidant largely abolished Nox4-induced ATF4 activation and ameliorated senescent cells in TG-BMECs.

Conclusions : Overexpression of Nox4 induces endothelial and vascular senescence in the retina in part through impairing mitochondrial biogenesis and activation of the ATF4/p16 pathway, which may contribute to the development of DR.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.