Abstract
Purpose :
Retinal pericyte loss is an early hallmark of diabetic retinopathy (DR); however, the mechanisms are not fully understood. Previously, we have shown that NADPH oxidase 4 (Nox4) is upregulated in DR, and Nox4 overexpression in endothelial cells (EC) leads to pericyte loss and capillary degeneration in mouse retinas. In this study, we investigated the mechanisms by which overexpressing Nox4 in ECs causes pericyte loss and its implication in DR.
Methods :
Human retinal endothelial cells (HRECs), brain microvascular endothelial cells (BMECs), and human retinal pericytes (HRPs) were used for in vitro experiments. Humanized EC-specific Nox4 transgenic (TG) and wild-type (WT) mice were used for in vivo study. Diabetes was induced by streptozotocin. Cellular senescence was examined by SA-β-gal assay, expression of senescence markers (P16, P21, and P53), and secretion of SASP factors. Mitochondrial dynamics were assessed by high-resolution single-molecule localization microscopy (SMLM). Cell viability was analyzed by crystal violet assay and apoptosis was examined by TUNEL assay.
Results :
Primary BMECs isolated from 2-month-diabetic mice demonstrated significantly increased senescence markers and Nox4 expression, suggesting an early upregulation of Nox4 associated with senescence in diabetic ECs. Overexpression of Nox4 by adenovirus induced a robust premature senescence phenotype in HRECs, which was abolished by treatment with catalase. Significantly increased senescence markers were also observed in BMECs and retinal microvessels of TG mice, accompanied by elevated SA-β-gal activity and increased DNA damage. Moreover, TG-BMECs displayed aberrantly elongated mitochondria associated with increased mitochondrial ROS and reduced mitochondrial respiration and produced higher levels of IL-1β and hydrogen peroxide (H2O2). Treatment of HRPs with H2O2 or conditioned medium from Nox4-overexpressing ECs reduced cell viability and increased apoptosis of HRPs.
Conclusions :
These results suggest that EC senescence occurs preceding pericyte loss in diabetes. Increased Nox4 expression induces premature senescence of ECs, which in turn leads to pericyte loss, in part through the paracrine actions of H2O2 and SASP factors. Thus, targeting EC senescence and restoring the normal Nox4 level may provide a new approach for protecting against pericyte loss and vasodegeneration in DR.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.