(VP078) NOX5 LINKS OXIDATIVE STRESS AND ENDOPLASMIC RETICULUM STRESS TO ENABLE VASCULAR SMOOTH MUSCLE CELL PHENOTYPIC SWITCHING IN HYPERTENSION

Background: Oxidative stress and endoplasmic reticulum (ER) stress through protein oxidation play a role in vascular dysfunction associated with hypertension. Nox5 is a major ROS-generating enzyme in in vascular smooth muscle cells (VSMCs) and is associated with an ER-perinuclear localization. Therefore, we investigated the global and oxidative proteomic profile and the role of Nox5-ER stress in VSMC signalling in human hypertension.

METHODS AND RESULTS: VSMC from resistance arteries from normotensive (NT) and hypertensive (HT) subjects were studied (n=5). Proteins were labelled with isobaric tandem mass tags and identified by liquid chromatography tandem mass spectrometry. The oxidative proteome was assessed using stable isotope-labelled iodoacetamide to target cysteine thiols. Nox5 compartmentalization, ER stress activation and protein expression were detected by western blotting. Nox5 and IRE1 silencing were performed by siRNA. The inflammatory and synthetic VSMC phenotype was assessed by measuring pro-inflammatory cytokines (IL-6, IL-8) and pro-collagen I in the culture media. Analysis of VSMC global proteome revealed upregulation of proteins involved in extracellular matrix organization (ECM), immune response and cell proliferation in HT subjects (fold change>1.5, p< 0.05), suggesting a synthetic and proinflammatory VSMC phenotype. Cysteine oxidation was increased in HT. We next assessed ER stress activation in VSMC. IRE1α (95%) and PERK arms (36%) of ER stress were increased in HT (p < 0.05). Nox5 expression was increased in HT in the ER/nuclear fraction (46%, p< 0.05). ER stress (4-PBA, 1mM) and IRE1 inhibition (STF083010, 60μM) decreased Nox5 expression in HT, while induction of ER stress (tunicamycin, 5μg/ml, 24h) increased Nox5 expression in cells from both groups (p < 0.05). Next, we investigated the effect of Nox5 and ER stress in the phenotypic switching of VSMC in HT. VSMCs from HT exhibit reduced expression of VSMC markers αSMA (90%), SM22 (83%) and MYOCD (50%), while KLF4 levels (240%) were augmented (p < 0.05). Additionally, expression of the proliferation marker, PCNA (217% vs NT, p< 0.05) and pro-collagen I (23.6±2 vs NT:13.2±0.3ng/ml, p< 0.05), and production of pro-inflammatory cytokines IL-6 (501±23 vs NT:121±6 pg/mL) and IL-8 (373±34.1 vs NT:262.5±24.6pg/mL, p< 0.05) were increased in HT. Nox5 and IRE1 silencing in VSMC from HT reduced PCNA expression, pro-collagen I release, baseline and LPS-induced IL-6 and IL-8 release (p < 0.05).

Conclusion: Our study identified proteomic changes related to vascular phenotype in human hypertension and highlighted redox-sensitive targets involved in human hypertension. We demonstrated an important role for Nox5-ER stress interplay in VSMC phenotypic switching, processes that contribute to vascular dysfunction in hypertension.