Introduction Diabetes is characterised by hyperglycaemia, which increases reactive oxygen species (ROS) production in endothelial cells (ECs). The consequent microvascular dysfunction drives progression of cardiovascular complications, such as cardiac remodelling and heart failure. NADPH oxidases are enzymes whose main function is to generate ROS and which are important in the development of cardiovascular disease. NOX4, in particular, is highly expressed in ECs and increased by hyperglycaemia. The aim of this project was therefore to identify the precise role of NOX4 in ECs under hyperglycaemic conditions with a specific role on ROS signalling and paracrine communication with fibroblasts, as key regulators of adverse cardiovascular remodelling in diabetes.

Methods Human aortic endothelial cells (HAoECs), with or without NOX4 siRNA knockdown (KD) were treated with normal (NG, 5.5 mM) or high glucose (HG, 25 mM) for up to 5 days. Cells were assessed for changes in mRNA and protein expression (real time (RT-PCR and Western blot, respectively; relative to GAPDH/HPRT/HSP90 and HPRT, respectively), and superoxide generation (DHE fluorescence), whilst conditioned media from NOX4-modified HAoECs with/without HG was incubated with NIH 3 T3 fibroblasts to interrogate effects on EC paracrine signalling.

Results After 5 days of HG treatment, HAoECs exhibited increased NOX4 mRNA expression (NG 1.01±0.06, HG 1.27±0.06; n=9, p<0.05), which was reflected at protein level. This was associated with upregulation of antioxidant (e.g. NRF2: NG 0.91±0.04, HG 1.22±0.04; n=6, p<0.05) and proinflammatory genes (e.g. IL-6: NG 1.03±0.13, HG 2.59±0.58; n=6, p<0.05) and elevated superoxide production after 2 but not 5 days (262±12 vs. 338±11 arbitrary units; n=4, p<0.05). Of note, NOX4 KD under HG conditions (25 mM for 2 days) increased mRNA expression of endogenous antioxidants (e.g. NRF2: NG 1.90±0.06, HG 2.21±0.06; SOD1: NG 0.89±0.01, HG 1.39±0.08; NQO1: NG 0.79±0.02, HG 1.025±0.06; n=3, p<0.05) whilst normalising the HG-induced increase in superoxide production. Moreover, conditioned media from HG-treated HAoECs increased TGF-β induced differentiation, which was ablated following NOX4 KD (e.g. α-SMA: scrambled control 1.31±0.04, NOX4 KD 0.94±0.07; CTGF: scrambled control 1.36±0.08, NOX4 KD 1.13±0.03; n=3, p<0.05).

Conclusions HG-induced NOX4 expression modulates ROS signalling in ECs, thereby altering paracrine regulation of fibroblast differentiation. EC-localised NOX4 is therefore likely to make a significant contribution to adverse cardiovascular remodelling in diabetes.