Abstract 068: Interplay Between Nox5 and Endoplasmic Reticulum Stress Regulates Vascular Signalling in Human Hypertension

Abstract

Nox5, a major ROS-generating oxidase in human vessels, regulates vascular contraction. We demonstrated an ER-perinuclear Nox5 localization and questioned the role of ER stress in Nox5 regulation. Vascular smooth muscle cells (VSMC) were isolated from small arteries from subcutaneous fat from normotensive (NT) and hypertensive (HT) subjects. Nox5 compartmentalization (cell fractionation); ROS generation (chemiluminescence); peroxiredoxin/DJ-1 oxidation, activation of ER stress and contractile signalling (IRE1α, Src, PKC, MLC phosphorylation; immunoblotting) and actin cytoskeleton organization (phalloidin staining) were assessed. In hypertension, ROS levels (139±27% vs NT, p<0.05), oxidation of peroxiredoxin (870.4±188.7% vs NT, p<0.05) and DJ-1 (125±34% vs NT, p<0.05) were increased. IRE1α phosphorylation was increased in the HT group (58±21% vs NT, p<0.05). ER stress inhibition (4-PBA, 1mM) reduced ROS levels in HT subjects (20±6% vs NT, p<0.05), suggesting association between ER and oxidative stress. Nox5 expression was increased in the HT group (103±23% vs NT, p<0.05) in a compartment specific manner: Nox5 levels were reduced in plasma membrane (45±7% vs NT, p<0.05), but increased in the ER/nuclear fraction (46±13% vs NT, p<0.05). IRE1 inhibition (STF083010, 60μM) decreased Nox5 expression in the HT group (65±5% vs Ctl, p<0.05), while induction of ER stress (tunicamycin, 5μg/ml, 24h) increased Nox5 expression in cells from both groups (p<0.05). To investigate the role of Nox5 on contractile signalling, cells were treated with mellitin (100nM), a Nox5 inhibitor. ROS generation and phosphorylation of c-Src, PKC and MLC 20 induced by Ang II were reduced by mellitin in both groups (p<0.05 vs Ctl). In contrast, silencing of p22phox increased ROS and activation of c-Src, PKC and MLC 20 in both groups, an effect blocked by mellitin (p<0.05 vs Ctl). VSMC from hypertensive subjects had increased number of stress fibres, an effect attenuated by mellitin. Our findings demonstrate that Nox5 is upregulated in a compartment specific manner and is regulated by ER stress in hypertension. Nox5 upregulation influences pro-contractile signalling and cytoskeleton reorganization in VSMC, processes that contribute to vascular dysfunction in hypertension.