Abstract
Purpose :
NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in endothelial cells (ECs). Sustained upregulation of Nox4 increases oxidative damage of retinal ECs contributing to vascular pathology in diabetic retinopathy (DR). The aim of this study is to evaluate the therapeutic potential of Nox4 inhibition in DR treatment using an inducible EC-specific Nox4 conditional knockout (cKO) mouse line.
Methods :
Inducible EC-specific Nox4 cKO mice were generated by crossing Nox4 floxed mice with Cdh5(PAC)-iCreERT2 mice. Induction of Cre expression was achieved by intraperitoneal injections of tamoxifen (TAM) at 1 month, 3 months, or 6 months after the onset of diabetes induced by streptozotocin. An mTmG reporter mouse line was used to confirm Cre-mediated KO in retinal ECs. Retinal vascular permeability was measured by FITC-dextran method at 6 months after diabetes. Retinal vascular changes were examined by fundus imaging, fluorescent angiography, and immunostaining in retinal whole mounts. Retinal morphology and thickness were assessed by optical coherence tomography (OCT) and histology at 9 months after diabetes. Primary brain microvascular ECs (BMECs) were isolated and cultured for in vitro study.
Results :
At 6 months after diabetes, inducible EC-Nox4 cKO mice showed significantly decreased retinal vascular permeability compared to diabetic wild type (WT) mice. No difference was observed in non-diabetic cKO and WT mice. At 9 months after diabetes, reduced retinal thickness and increased number of acellular capillaries in the retina were observed in diabetic WT mice and to a significantly lesser extent in diabetic cKO mice, suggesting that deletion of Nox4 at the early stage of DR can successfully prevent vascular and retinal degeneration. Preliminary results show that induction of Nox4 deletion in ECs at 6 months post-diabetes also led to reduced acellular capillary formation and attenuated retinal thinning at 9 months after diabetes; however, the differences did not reach significance. Moreover, BMECs isolated from EC-Nox4 cKO mice showed significantly preserved expression of endothelial tight junction proteins ZO-1, VE-Cadherin, and CD31 under hypoxia condition.
Conclusions :
Our results suggest that targeting EC-Nox4 after the onset of diabetes can prevent the development and progression of DR by improving the vascular permeability and retinal degeneration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.