Exploring the roles of NADPH oxidase (NOX) isoforms, NOX1 and NOX4 in the accelerated development and progression of atherosclerosis in diabetes.

2017-02-24T02:03:09Z (GMT) by Di Marco, Elyse
Chronic hyperglycaemia and oxidative stress in diabetes trigger sustained vascular inflammation as well as pronounced aberrations in cellular structure and function, which drive atherosclerosis A major source of reactive oxygen species (ROS) in the vasculature are NADPH Oxidase (NOX) enzymes. The roles of different NOX isoforms, namely NOX1 and NOX4, in immune and vascular cell pathophysiology remain to be fully elucidated. Furthermore, the relative contribution of NOX1 and NOX4 to early versus advanced atherosclerosis mediated by short and long-term exposure to the diabetic milieu, respectively, is unclear. The unifying aim of this thesis is to improve the current knowledge of NOX1 and NOX4 in pro- and anti-atherosclerotic processes in diabetes. Atherosclerosis-prone Apoe-/- mice with or without genetic deletion of NOX1 or NOX4 were rendered diabetic using streptozotocin (STZ) and followed for 10- and 20-weeks. Concurrently, a subgroup of Apoe-/- mice were administered the NOX1-NOX4 dual pharmacological inhibitor, GKT137831 (60 mg/kg/day). The development and complexity of atherosclerotic lesions across the aorta were examined histologically. Pathological signalling pathways were analysed at the gene and protein level. Oxidative stress parameters were assessed qualitatively and quantitatively. Mechanistic studies of NOX-mediated cellular pathology were performed in human aortic endothelial cells (HAECs) and primary mouse aortic smooth muscle cells (SMCs). The 10-week studies clearly demonstrated the pro-inflammatory and pro-atherogenic properties of NOX-derived ROS in the aortic sinus and aortic arch of diabetic Apoe-/- mice. Both NOX1 and NOX4 were shown to play important roles in diabetes-associated atherosclerosis (DAA) and protein oxidation in the aortic sinus. We identified an important role for NOX4 in recruitment of T cells to the atherosclerotic lesion, CD4+ T cell activation in the draining mediastinal lymph nodes and the induction of pro-inflammatory cytokine gene expression. Deletion of NOX1 attenuated macrophage infiltration into the aortic sinus. In the aortic arch and thoraco-abdominal regions, we identified NOX1- but not NOX4-derived ROS as the important mediators of inflammation and fibrosis following 10-weeks of diabetes. Investigations in HAECs treated with siRNA against the NOX1 transcript showed reduced oxidative stress, pro-inflammatory and pro-fibrotic gene expression, that were not observed in NOX4 siRNA- treated HAECs. At the 20-week study endpoint, we demonstrated a protective role for NOX4-derived ROS in DAA. Deletion of NOX4 exacerbated atherosclerotic plaque size in diabetic Apoe-/- mice via enhanced vascular inflammation, fibrosis and SMC de-differentiation. Conversely, deletion of NOX1 attenuated atherosclerosis in Apoe-/- mice exposed to 20-weeks of diabetes, in part via reduced oxidative stress and macrophage recruitment. Aortic SMCs isolated from NOX4-deficient Apoe-/- mice showed decreased expression of genes associated with contractility, proliferation and fibrosis, which were restored by inhibition of PDGF or silencing of NOX1. Collectively, these findings revealed distinct roles for NOX1 and NOX4-derived ROS in pro- inflammatory and pro-fibrotic pathways at different stages of atherosclerosis mediated by diabetes, identifying important considerations for the future development and application of NOX-based therapies for DAA.