Endothelial cell Nox4 is an important regulator of cardiac ROS signalling and adverse remodelling in experimental diabetes

Introduction: Hyperglycaemia promotes inappropriate reactive oxygen species (ROS) signalling and adverse cardiac remodelling. NADPH oxidases are major sources of cardiovascular ROS, whose vascular expression, particularly in endothelial cell (ECs), is upregulated in the diabetic heart. Our aim was to identify the specific role of EC Nox4 in cardiac remodelling associated with experimental diabetes. Results: Diabetes was induced in EC-specific Nox4-overexpressing (Nox4+/-) and wildtype (Nox4WT) mice by streptozotocin (STZ) injection (n=9-11). After 6 months, echocardiography indicated diastolic dysfunction in Nox4WT diabetic mice (E/A: 1.94±0.10 vs control 1.60±0.13; P<0.05) but not Nox4+/- diabetic mice (E/A: 1.55±0.06 vs control 1.54±0.09, P=NS). Similarly, Nox4WT but not Nox4+/- diabetic mice demonstrated increased cardiac fibrosis (Picrosirius red staining) and profibrotic gene expression (qRT-PCR) versus controls. Interestingly, Nox4+/- diabetic mice exhibited markedly elevated antioxidant gene expression (e.g. SOD: Nox4+/- 3.12±0.43, Nox4WT 1.75±0.32; P<0.05), associated with decreased ROS production (DCF fluorescence), which may at least partly underlie protection against diastolic dysfunction. Consistent with these data, parallel studies in human aortic ECs (HAoECs; n=3) treated with high glucose (HG, 25mmol/L) for 48h showed that Nox4 overexpression (OE) increased antioxidant gene expression (e.g. catalase: HG Nox4OE 2.85±0.40, HG empty vector [EV] control 1.20±0.11; P<0.05), whilst conditioned media from Nox4OE HAoECs promoted TGFβ-induced 3T3 fibroblast differentiation (e.g. α-smooth muscle actin: HG Nox4OE 1.39±0.16, HG EV 0.88±0.04; P<0.05). In contrast, Nox4 knockdown reduced HG-induced antioxidant gene expression, which was associated with normalisation of superoxide production (DHE fluorescence) and fibroblast differentiation. Conclusion: EC Nox4 is an important regulator of ROS signalling in experimental diabetes which may determine progression of adverse cardiac fibrosis and remodelling. Further work is required to elucidate specific underlying mechanisms.