NOX5 AND VASCULAR SIGNALLING IN HUMAN HYPERTENSION

Abstract

Objective: Nox5, a key ROS-generating oxidase in human vessels, regulates vascular contraction and is implicated in vascular damage. However molecular mechanisms regulating vascular Nox5 are elusive. We questioned the role of Nox5 in vascular smooth muscle cell signalling in hypertension. Design and method: Intact small arteries and vascular smooth muscle cells (VSMC) isolated from small arteries from normotensive (NT) and hypertensive (HT) subjects were studied. Nox5 expression and phosphorylation (western blot, immunoprecipitation); ROS generation (chemiluminescence); protein oxidation (oxidised peroxiredoxin/DJ-1), activation of signalling pathways (Src, PKC, MLC phosphorylation; immunoblotting), and actin cytoskeletal organization (phalloidin staining) were assessed in the absence and presence of Nox5 inhibition (mellitin, 100 nM). To exclude Nox1–4 effects, we used p22phox silenced VSMCs (siRNA). Results: Nox5 expression was increased in intact arteries from HT subjects (70 + 26.79% vs NT, p < 0.05). In VSMC, ROS levels (139 ± 27% vs NT, p < 0.05), and oxidation of peroxiredoxin (201.8 + 40.34% vs NT, p < 0.05) and DJ-1 (71.7 + 16.51% vs NT, p < 0.05) were increased in HT. Nox5 expression (103 ± 23% vs NT, p < 0.05) and phosphorylation (77 + 17.93% vs NT, p < 0.05) were increased in the HT group. Nox5 inhibition reduced Ang II-induced ROS generation in both groups (p < 0.05 vs Ctl). In contrast, p22phox silencing increased ROS in both groups, an effect blocked by mellitin (p < 0.05 vs Ctl). Nox5 associates with c-Src. Treatment with a Src inhibitor (PP2, 10 μM) did not reduce Nox5 phosphorylation. Furthermore, PP2 failed to reduce AngII-induced ROS generation in p22phox-silenced VSMCs. Nox5 inhibition reduced Ang II-induced c-Src activation (p < 0.05 vs Ctl), suggesting that Nox5 is upstream of c-Src. Next, we investigated the role of Nox5 in vascular signaling. In cells from hypertensive subjects, p22phox silencing was associated with sustained Ang II-induced PKC and MLC20 phosphorylation, an effect blocked by mellitin and PP2 (p < 0.05 vs Ctl). VSMC from hypertensive subjects had an increased number of stress fibres, which was attenuated by mellitin and PP2. Conclusions: Our findings demonstrate that in human hypertension Nox5 is upregulated. This is associated with activation of c-Src, increased redox-sensitive signalling and VSMC cytoskeletal reorganisation. We define a novel Nox5-ROS-c-Src signalling pathway that may play a role in vascular remodelling in hypertension.