The microvascular endothelial redoxome established through tandem mass tag mass spectrometry

Redox signalling plays an important role in endothelial cell (EC) physiology and pathophysiology. Proteins sense redox signals via cysteine thiol groups. A common oxidative post-translational modification (oxPTM) on cysteine thiols is S-glutathionylation which is reversed to a free thiol state by glutaredoxin (Glrx). OxPTMs alter protein function, location and stability. Identifying which proteins undergo modification will help determine the role of redox signalling in EC function. A proteome-wide screen of human cardiac microvascular endothelial cells identified redox-sensitive proteins involved in vascular signalling mechanisms. Methods Human microvascular endothelial cells (HCMVEC, Lonza) were exposed to VEGF (50ng/ml, 24h) or hypoxia (0.5% O2, 24h) with adenoviral (ad) Glrx (Vectorlabs) overexpression or adLacZ (control). A tandem mass tag mass spectrometry system (TMT) coupled with a thiol-switch technique was used to quantify changes in redox sensitive thiol modifications. Protein lysates were treated with MMTS to alkylate unmodified thiols. Iodo-TMT six-plex probes were tagged to redox-sensitive sites after reversal of oxPTMs by DTT. Samples were pooled and processed by nLC-MS/MS. The abundance of each peptide in different conditions was compared with either adGlrx or adLacZ (control) expression to provide a ratio of changes in redox modifications. Results Iodo-TMT analysis revealed 113 unique thiol modifications identified on 78 different proteins using a ±1.5-fold threshold in a given treatment. Additionally, 44 modifications in 33 proteins were present in at least 2 different conditions, namely Glrx under VEGF and hypoxic conditions. A STRING interaction network identified clusters of 10 proteins involved in organonitrogen synthesis and 6 proteins in angiogenesis. Jagged-1 involved in the regulation of angiogenic sprouting through the Notch pathway was established as a target of redox signalling. Identified redox sensitive cysteines were found in extracellular EFG1 and the calcium binding EGF12 domains. Seven different In Silico programs (including MutationTaster, PolyPhen-2 and PANTHER) predicting the impact of substitution mutations indicated a functional affect for these redox sensitive sites, demonstrating the importance of these residues. Conclusion A non-biased proteomics approach identified novel thiol modifications on proteins involved in microvascular function. Future work will demonstrate the impact of these redox-sensitive thiol modifications on microvascular function to provide a better understanding of redox signalling in protein function and disease. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): MRC-DTP,European Union's Horizon 2020 research and innovation programme.

Patient Endothelial Colony-Forming Cells to Model Coronary Artery Disease Susceptibility and Unravel the Role of Dysregulated Mitochondrial Redox Signalling

Mechanisms involved in the individual susceptibility to atherosclerotic coronary artery disease (CAD) beyond traditional risk factors are poorly understood. Here, we describe the utility of cultured patient-derived endothelial colony-forming cells (ECFCs) in examining novel mechanisms of CAD susceptibility, particularly the role of dysregulated redox signalling. ECFCs were selectively cultured from peripheral blood mononuclear cells from 828 patients from the BioHEART-CT cohort, each with corresponding demographic, clinical and CT coronary angiographic imaging data. Spontaneous growth occurred in 178 (21.5%) patients and was more common in patients with hypertension (OR 1.45 (95% CI 1.03–2.02), p = 0.031), and less likely in patients with obesity (OR 0.62 [95% CI 0.40–0.95], p = 0.027) or obstructive CAD (stenosis > 50%) (OR 0.60 [95% CI 0.38–0.95], p = 0.027). ECFCs from patients with CAD had higher mitochondrial production of superoxide (O2−–MitoSOX assay). The latter was strongly correlated with the severity of CAD as measured by either coronary artery calcium score (R2 = 0.46; p = 0.0051) or Gensini Score (R2 = 0.67; p = 0.0002). Patient-derived ECFCs were successfully cultured in 3D culture pulsatile mini-vessels. Patient-derived ECFCs can provide a novel resource for discovering mechanisms of CAD disease susceptibility, particularly in relation to mitochondrial redox signalling.