Abstract
Purpose :
Oxidative stress resulting from excessive reactive oxygen species (ROS) generation is a major cause of angiogenic progenitor dysfunction that contributes to the development of persistent vascular damage in diabetic retinopathy (DR). Main sources of ROS in blood vessels include mitochondria and NADPH oxidases. However, the expression and role of NADPH oxidases in diabetic angiogenic progenitors and their implication in DR remain poorly understood. Herein, we investigate the role of NADPH oxidase 4 (Nox4) in diabetic angiogenic progenitor dysfunction and DR.
Methods :
Diabetes was induced in Tie2-cre mediated Nox4 conditional KO (cKO) mice and wild type (WT) mice. Expression of Nox isoforms (Nox1, Nox2, and Nox4) was examined in bone marrow-derived endothelial outgrowth cells (EOCs) at 1, 3, 6, or 9 months after the induction of diabetes. Cell proliferation, colony formation, apoptosis, senescence, migration, and ROS generation were examined in EOCs from 6-month diabetic Nox4 cKO and WT mice. Signaling pathways of oxidative stress, ER stress, and expression of integrins were investigated.
Results :
At 6 and 9 months after diabetes, Nox4 expression in EOCs was significantly increased, while Nox2 expression in EOCs was increased only after 9 months diabetes and no change was observed in Nox1 expression at any time points. In addition, ROS levels were significantly increased in 6 month-diabetic EOCs and the increase was largely abolished in Nox4 cKO cells. Compared to non-diabetic controls, EOCs derived from 6-month diabetic WT mice demonstrated reduced proliferation and colony formation, enhanced apoptosis, increased senescence, and impaired migration in response to VEGF, all of which were significantly improved in EOCs from diabetic Nox4 cKO mice. Mechanically, Nox4 deletion remarkably reduced endoplasmic reticulum (ER) stress and alleviated CHOP and caspase-3 activation in diabetic EOCs. Furthermore, downregulation of Nox4 or inhibition of ER stress significantly alleviated diabetes-induced reduction of integrin b1 improving mobilization of EOCs.
Conclusions :
Our results suggest that the aberrant upregulation of Nox4 in diabetic EOCs resulting in increased ROS, enhanced ER stress and disrupted integrin expression may play an important role in angiogenic progenitor dysfunction and DR development.
This is a 2021 ARVO Annual Meeting abstract.