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Sarah Xin Zhang, Xixiang Tang, Hanna E Abboud, Yanming Chen, Joshua Jianxin Wang; Endothelial Nox4 Deletion Mitigates Retinal Vascular Abnormalities in Diabetes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2731.
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© ARVO (1962-2015); The Authors (2016-present)
NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in endothelial cells (ECs), but its roles in regulation of endothelial function and vascular injury during diabetes remain controversial. Using EC-specific Nox4 knockout (Nox4EC-KO) mice, in which Nox4 was conditionally deleted in ECs, and EC-specific Nox4 transgenic (Nox4EC-Tg) mice that overexpress human Nox4 in ECs, we examined the function of endothelial Nox4 in the development of vascular pathologies in diabetic retinopathy.
Nox4EC-KO mice and Nox4EC-Tg mice were generated by crossing Nox4 floxed mice or lox-Stop-lox-human Nox4 Tg mice with Tie2-cre mice. Diabetes was induced with streptozotocin in mice at age of 8 weeks. Retinal vascular morphology and vascular permeability were examined by fluorescence angiography, immunostaining, and FITC-dextran methods. Retinal acellular capillary formation was assessed by double staining of retinal whole mounts with anti-Collagen IV antibody and Alexa Fluor 594-conjugated isolectin GS B4. ROS generation and apoptosis were examined in mouse brain microvascular ECs (BMECs).
Successful deletion or overexpression of Nox4 in ECs was confirmed by measuring Nox4 mRNA and protein levels as well as by assessing ROS in BMECs isolated from Nox4EC-KO mice and Nox4EC-Tg mice. At 3 months after hyperglycemia onset, diabetic WT mice demonstrated a 5-fold increase in retinal vascular permeability compared to non-diabetic controls and this increase was blunted in Nox4EC-KO mice. At 6 months after diabetes, a significant increase in acellular capillary formation was observed in diabetic WT mice but not in Nox4EC-KO mice. There was no significant difference in vascular permeability and acellular capillaries between non-diabetic Nox4EC-KO mice and non-diabetic WT mice. In contrast, Nox4EC-Tg mice at ages of 10 – 12 months exhibited increased tortuosity of retinal blood vessels, focal vascular leakage, and acellular capillary formation, compared to age-matched WT mice. In vitro study confirmed enhanced apoptosis and cellular senescence in BMECs derived from Nox4EC-Tg mice.
Our study provides strong in vivo evidence that Nox4 upregulation in retinal ECs is essential for diabetes-induced vascular leakage and acellular capillary formation. Inhibition of Nox4 overactivation in ECs may provide a new approach for the treatment of diabetic vasculopathy in the retina.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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