Abstract P502: Vascular Tissue Proteomics Enables Detection of Cell Type-specific Changes in Target Expression

Introduction: Identification of robust targets from proteomic studies in cardiovascular tissue may prove challenging. Using an angiotensin II (Ang II)-infusion mouse model, we performed a proteomics study in isolated thoracic aortas. Changes in proteins related to cardiovascular pathophysiology were identified and candidate targets selected for validation via traditional techniques. Methods and Results: C57 black/6 mice were infused with Ang II (24 μg/kg/hour) via osmotic minipump for 6 weeks. Elastic Van Gieson staining demonstrated significantly increased medial area in aortas from Ang II-infused mice compared to water-infused control mice ( P <0.005 ), indicating Ang II-induced remodelling. Nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) revealed a 1.28 fold increase in galectin-3 (LGALS3) expression in vessels from Ang II-infused mice as compared to controls. LGALS3, a β-galactoside binding lectin, is a well known marker of cardiovascular disease reported to play a role in Ang II-induced cardiac remodelling. Immunohistochemical (IHC) staining showed increased LGALS3 expression throughout the vessel wall and particularly in the endothelial layer (quantification using Image J Fiji software: 110.4 ± 1.37 vs 120.5 ± 2.6 arbitrary units; P =0.005) in Ang II-infused mice compared to controls. Human primary endothelial cells (ECs) were isolated from saphenous veins of patients undergoing coronary artery bypass graft surgery and translational studies performed. LGALS3 /LGALS3 expression was detected at both mRNA and protein level by qRT-PCR and immunoblotting respectively. Acute stimulation of ECs with Ang II (200nM for 24 hours) failed to upregulate LGALS3 /LGALS3 expression suggesting that the increased endothelial expression observed in vivo is due to chronic infusion of Ang II. Conclusions: We have successfully validated the Ang II-induced increase in LGALS3 identified via vascular tissue proteomics. Despite the use of homogenised whole aortic tissue, nano LC-MS/MS proved sensitive enough to detect elevated expression of a candidate protein that is predominantly expressed in the endothelium. Tissue proteomics can detect changes in expression specific to a single cell type.