Abstract P342: Vascular Protein Oxidation and Redox Proteomics in Hypertension

Abstract

In hypertension (HTN) mechanisms whereby protein oxidation regulates vascular function remain unclear. We hypothesise that increased ROS promote a shift of oxidative post-translational protein modifications from reversible to irreversible forms, leading to aberrant redox signalling and vascular injury. VSMC from mesenteric arteries of normotensive (WKY) and hypertensive (SHRSP) rats were stimulated with Ang II (10 -7 M) in the presence/absence of PEG-catalase (1000U/ml) or tempol (10 -5 M). Protein carbonylation was assessed by oxyblot and protein sulfenylation by DCP-Rho1 fluorescent probe. Protein tyrosine phosphatase (PTP)-oxidation, peroxiredoxin hyperoxidation (PRXSO3), γH2AX, Bcl2 levels were assessed by immunoblotting. DiGE and CyDye labelling screened for reversibly oxidised thiol proteome. Irreversible carbonylation and PRXSO3 were increased in SHRSP (fold change (FC)=1.29 and 2.77, p<0.05). Ang II-stimulation did not alter carbonylation levels. Reversible sulfenylation and thiol-proteome oxidation were reduced in SHRSP (FC=-1.18, p<0.05 and 13.6% (253 spots)). Ang II-treatment increased sulfenylation in WKY (FC=1.08, p<0.05) and SHRSP (FC=1.23, p<0.001); an effect inhibited by catalase. Reversible PTP oxidation was increased in WKY and SHRSP (FC=1.92 and 2.42, p<0.05), versus irreversible levels. Irreversible PTP oxidation tended to be higher in SHRSP. Ang II increased reversible PTP oxidation only in WKY (FC=1.27, p<0.05) and it was prevented by tempol. Ang II-stimulation increased protein levels of γH2AX (DNA damage) (FC=1.76, p<0.05) and Bcl2 (anti-apoptotic) (FC=2, p<0.05) in WKY. Proteomic data, filtered for FC>2, detected 1777 spots with 21% being differentially oxidised between WKY and SHRSP. Candidate proteins differentially oxidized between WKY and SHRSP include annexin A1 (-2.29) and galectin-1 (2.83). These results demonstrate altered redox status in HTN characterised by increased protein hyperoxidation and decreased reversible oxidation, in combination with decreased anti-oxidant capacity. Moreover, our findings identify novel candidate oxidized proteins implicated in VSMC motility, proliferation and signalling which may contribute to oxidative vascular injury in HTN.