Proteomics Characterization of Extracellular Space Components in the Human Aorta

Abstract

The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis.

Atypical Hemolytic Uremic Syndrome-Associated Variants and Autoantibodies Impair Binding of Factor H and Factor H-Related Protein 1 to Pentraxin 3

“…4). Although the binding of CFH to ECM was not of high affinity under our experimental conditions, it is likely relevant in vivo (49,50). One also has to consider that a relatively modest change in the concentration of regulators can have a decisive effect on complement activation because of the exponential characteristic of the activation steps.…”

Romosozumab or Alendronate for Fracture Prevention in Women with Osteoporosis

“…Sclerostin is constitutively expressed in the aorta [16][17][18] and up-regulated in foci of vascular and valvular calcification. [19][20][21][22] The function of sclerostin in the vasculature is unknown.…”

Extracellular matrix proteins and the liver-lung axis in disease.

“…For this study, a previously described 3-step ECM fractionation method was adapted for use with mouse liver tissue (95)(96)(97). The ECM isolation method was performed on hepatic tissue of from and a model of ASH which does not cause significant fibrosis.…”

“…Furthermore, proteomic-based studies in other organs haves demonstrated that the matrisome responsds dynamically in composition after insult (95)(96)(97).…”

“…However, recent studies have shown that multiple ECM proteins contribute to fibrogenesis (48), and that the hepatic ECM is altered prior to fibrogenesis (28). Therefore the goal of Chapter V was to adapt a sequential protein extraction method developed for cardiovascular research (95)(96)(97) to facilitate LC-MS analysis of global changes in the hepatic matrisome after chronic alcohol exposure. Such an approach could discover new proteins that are changed by insults.…”