Project 5

Vascular redox signalling and protein oxidation in hypertension - focus on inflammation and fibrosis

Objectives:
Hypertension-associated target organ damage is the major pathophysiological process that underlies cardiovascular disease, including heart failure, kidney failure and stroke. Fundamental to these events is vascular dysfunction and arterial remodelling, driven in large part by oxidative stress, which leads to inflammation and fibrosis. Reactive oxygen species (ROS) are key mediators of signalling pathways that underlie endothelial dysfunction and vascular injury. Elucidating how ROS modify proteins, which proteins are differentially oxidized in hypertensive vascular disease and what fraction is reversibly versus irreversibly oxidised are crucial aspects of redox biology, because ultimately it is these processes that determine cell fate and function. The main goal is to characterise the vascular oxidative proteome and redox signalling in hypertension, focusing on vascular dysfunction and end organ damage.

Methodologies:
Studies will be performed in isolated vessels and cultured vascular smooth muscle cells (VSMC) from Ang II-dependent hypertensive mice (LinA3 mice, expressing human renin) and wild-type controls. We will also use unique tissue from clinically phenotyped normotensive and hypertensive subjects (ethics approval in place). Vascular structure and function will be assessed by vessel myography, advanced microscopy (Intelligent Imaging), omics, biochemical, molecular, redox and vascular biology approaches in the absence and presence of pro-oxidants (H2O2; KO2-), anti-oxidants (tempol, NAC) and Nox inhibitors. All cell-based studies are performed in 6–8 different samples. Novel methods are used to assess protein oxidation. Oxidative vascular proteomics will be performed in VSMCs and intact vessels using quantitative analysis of the reversibly oxidized Cys proteome where free thiols are irreversibly blocked by reduction and labelling of modified thiols with iodoTMT™ isobaric labels. Iodo TMT tags are cysteine-specific and enable up to six different samples to be analysed and quantified by LC-MS/MS (Attoquant). This approach allows evaluation of transient Cys oxidation and identification of differentially oxidised proteins in normal and hypertensive conditions (milestone M20). Post-translational oxidative modification (i). Reversible oxidation. BCN-E-BCN and DCP-Bio probes are used to assess sulfenylation and affinity capture of sulfenylated proteins is performed with high capacity streptavidin beads. (ii) Oxidation of vascular signalling proteins. Sulfenylated proteins are captured by DCP-Bio1 and probed for cytoskeletal and signalling proteins. iii) Irreversible oxidation. Protein carbonylation is assessed using the oxyblot system. Redox-dependent signalling using immunostudies will evaluate total, phosphorylated and oxidised forms of pro-contractile, pro-inflammatory and pro-fibrotic signalling. Protein oxidation damage will also be assessed in renal and cardiac tissue.

Significance:
This comprehensive study will identify an integrative molecular and cellular framework that defines the vascular phenotype in hypertension, providing insights into mechanisms of disease and elucidation of novel therapeutic targets.

Secondments:
Planned academic secondments include Dr Ana Briones; Prof. Maria Soledad Fernandez-Alfonso (Madrid); Prof. Pierre Boutouyrie (Paris); Dr Koen Reesink (Maastricht). Planned industry secondments include Attoquant (Vienna); Intelligent Imaging Innovations (Germany); TMC Datascience (Netherlands).