Vascular proteomics in metabolic and cardiovascular diseases

Abstract

The vasculature is essential for proper organ function. Many pathologies are directly and indirectly related to vascular dysfunction, which causes significant morbidity and mortality. A common pathophysiological feature of diseased vessels is extracellular matrix (ECM) remodelling. Analysing the protein composition of the ECM by conventional antibody-based techniques is challenging; alternative splicing or post-translational modifications, such as glycosylation, can mask epitopes required for antibody recognition. By contrast, proteomic analysis by mass spectrometry enables the study of proteins without the constraints of antibodies. Recent advances in proteomic techniques make it feasible to characterize the composition of the vascular ECM and its remodelling in disease. These developments may lead to the discovery of novel prognostic and diagnostic markers. Thus, proteomics holds potential for identifying ECM signatures to monitor vascular disease processes. Furthermore, a better understanding of the ECM remodelling processes in the vasculature might make ECM-associated proteins more attractive targets for drug discovery efforts. In this review, we will summarize the role of the ECM in the vasculature. Then, we will describe the challenges associated with studying the intricate network of ECM proteins and the current proteomic strategies to analyse the vascular ECM in metabolic and cardiovascular diseases.

Keywords: apolipoproteins; atherosclerosis; diabetes; proteomics; restenosis; vascular biology.

Figure 1

Proteomics workflow targeting the extracellular matrix (ECM). Biochemical subfractionation allows enrichment of ECM proteins. Vascular tissues of human origin or those obtained from animal models of cardiovascular disease are initially incubated in 0.5 mol L⁻¹ NaCl to extract proteases, growth factors and newly synthesized ECM proteins. A subsequent decellularization step is performed with a buffer containing sodium dodecyl sulphate (SDS) before integral, polymeric ECM proteins are extracted with guanidine hydrochloride (GuHCl). Proteins obtained after sequential extraction are digested into peptides before liquid chromatography–tandem mass spectrometry analysis. The histological panel (haematoxylin and eosin staining) demonstrates the efficacy of SDS in removing cellular components before extraction of ECM proteins in human aortas. L, lumen; I, intima; M, media. Adapted from Didangelos et al. 18.

Figure 2

Proline hydroxylation of collagens. Proline hydroxylation (i.e. 4‐hydroxyproline and 3‐hydroxyproline) is a common modification that confers stability to collagen triple helices. (a) Inclusion of this variable modification in the search parameters dramatically improves identification and quantification of collagen peptides. The example shown is a peptide spanning amino acid positions 935–958 of human collagen 1, α‐1 chain (CO1A1). (b) CO1A1 protein sequence coverage is also improved ~10‐fold after searching liquid chromatography–tandem mass spectrometry data from human aortic specimen and including this variable PTM. Pro, proline.

Figure 3

Glycosylation of extracellular matrix (ECM) proteins. More than 90% of ECM proteins are glycosylated, which can affect protein identification with antibodies. (a) Attachment of N‐linked oligosaccharides to ECM proteins occurs at specific consensus sequences. Biglycan is an example of a heavily glycosylated ECM protein. (b) Removal of glycosaminoglycans (GAGs) and small oligosaccharides or removal of GAGs alone affects protein migration by gel electrophoresis. Biglycan contains GAGs and oligosaccharides, and mimecan contains only N‐linked oligosaccharides, whilst galectin‐1 is a nonglycosylated ECM protein. The aim of glycoproteomics is the identification of the protein core and the attached glycan.