Abstract 020: Nox5 Regulates Vascular Smooth Muscle Cell De-differentiation In Human Hypertension

Abstract

Nox5 is an important source of reactive oxygen species and aberrant redox-sensitive signalling in vascular smooth muscle cells (VSMC) in human hypertension. We aimed to characterize the VSMC proteomic profile and investigate the effects of Nox5-derived ROS on VSMC phenotype in human hypertension. VSMC from resistance arteries from normotensive (NT) and hypertensive (HT) subjects were studied. 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 silencing was performed by siRNA. Protein expression was detected by western blotting. Pro-inflammatory cytokines (IL-6, IL-8) and pro-collagen I was measured by ELISA in the culture media. Proteomic analysis identified 207 proteins upregulated in HT subjects (fold change>1.5, p<0.05). Gene ontology enrichment analysis showed proteins upregulated in HT were involved in extracellular matrix (ECM) organization, immune response and cell proliferation. ECM proteins (COL1A1, COL9A1, COL10A1, FBN1, FBLN1) and proteins related to interferon and IL-1β pathways (IFIT1, IFIT2, IFIT3, MX1, MX2, ABCA1, ABCA2, IL1RAP, CD36, ICAM1) were increased in cells from HT subjects. The VSMC oxidative proteome analysis identified 88 cysteine-containing peptides highly oxidized in HT (fold change>1.5, p<0.05), including COL11A1, COL16A1, FBLN1 and FBLN2. VSMCs from HT exhibit increased expression of the proliferation marker, PCNA (0.162±0.3 vs NT:0.051±0.04RFU, p<0.05) and pro-collagen I (23.6±2 vs NT:13.2±0.3ng/ml, p<0.05). Production of pro-inflammatory cytokines IL-6 (501.8±23.6 vs NT:121.7±6.4pg/mL) and IL-8 (373.6±34.1 vs NT:262.5±24.6pg/mL, p<0.05) were increased in HT. Nox5 silencing in VSMC from HT subjects reduced PCNA expression(43%), pro-collagen I release (8%), baseline and LPS-induced IL-6 (30% baseline, 43% LPS-induced) and IL-8 (21% baseline, 23% LPS-induced) release (p<0.05). We provide new insights into the proteomic changes related to vascular phenotype in hypertension and demonstrated that Nox5 plays an important role in VSMC phenotypic switching associated with vascular dysfunction in hypertension.