Glycoproteomic Analysis of the Aortic Extracellular Matrix in Marfan Patients.

OBJECTIVE: Marfan syndrome (MFS) is caused by mutations in FBN1 (fibrillin-1), an extracellular matrix (ECM) component, which is modified post-translationally by glycosylation. This study aimed to characterize the glycoproteome of the aortic ECM from patients with MFS and relate it to aortopathy. Approach and Results: ECM extracts of aneurysmal ascending aortic tissue from patients with and without MFS were enriched for glycopeptides. Direct N-glycopeptide analysis by mass spectrometry identified 141 glycoforms from 47 glycosites within 35 glycoproteins in the human aortic ECM. Notably, MFAP4 (microfibril-associated glycoprotein 4) showed increased and more diverse N-glycosylation in patients with MFS compared with control patients. MFAP4 mRNA levels were markedly higher in MFS aortic tissue. MFAP4 protein levels were also increased at the predilection (convexity) site for ascending aorta aneurysm in bicuspid aortic valve patients, preceding aortic dilatation. In human aortic smooth muscle cells, MFAP4 mRNA expression was induced by TGF (transforming growth factor)-β1 whereas siRNA knockdown of MFAP4 decreased FBN1 but increased elastin expression. These ECM changes were accompanied by differential gene expression and protein abundance of proteases from ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family and their proteoglycan substrates, respectively. Finally, high plasma MFAP4 concentrations in patients with MFS were associated with a lower thoracic descending aorta distensibility and greater incidence of type B aortic dissection during 68 months follow-up. CONCLUSIONS: Our glycoproteomics analysis revealed that MFAP4 glycosylation is enhanced, as well as its expression during the advanced, aneurysmal stages of MFS compared with control aneurysms from patients without MFS

Glycoproteomic analysis of the secretome of human endothelial cells

Previous proteomics studies have partially unravelled the complexity of endothelial protein secretion, but did not investigate glycosylation, a key modification on secreted and membrane proteins for cell communication. In this study, human umbilical vein endothelial cells were kept in serum-free medium before activation by phorbol 12-myristate 13-acetate, a commonly used secretagogue that induces exocytosis of endothelial vesicles. Apart from 123 secreted proteins, the secretome was particularly rich in membrane proteins. Glycopeptides were enriched by ZIC-HILIC resins and were either treated with PNGase F and H(2)(18)O or directly analysed using a recently developed HCD-ETD workflow for a hybrid linear ion trap-orbitrap MS. After deglycosylation with PNGase F in the presence of H(2)(18)O, 123 unique peptides displayed (18)O-deamidation of asparagine, corresponding to 86 proteins with a total of 121 glycosylation sites. Direct glycopeptides analysis by HCD-ETD identified 131 glycopeptides from 59 proteins and 118 glycosylation sites, of which 41 were known, 51 were predicted and 26 were novel. Two methods were compared: alternating HCD-ETD and HCD-product dependent ETD. The former detected predominantly high intensity, multiply charged glycopeptides while the latter preferentially selected precursors with complex / hybrid glycans for fragmentation. Validation was performed by glycoprotein enrichment and analysing the input, the flow through and the bound fraction. This study represents the most comprehensive characterisation of endothelial protein secretion to date and demonstrates the potential of new HCD-ETD workflows for determining the glycosylation status in complex biological samples

Proteomic characterization of human early pro-angiogenic cells

Early pro-angiogenic cells (EPCs) have been shown to be involved in neovascularization, angiogenesis and re-endothelialization and cathepsin L inhibition blunted their pro-angiogenic effect. In the present study, we have analysed and mapped the proteome and secretome of human EPCs, utilizing a combination of difference in-gel electrophoresis (DIGE) and shotgun proteomics. A population of 206 protein spots were analysed, with 171 being identified in the cellular proteome of EPCs. 82 proteins were identified in their conditioned medium, including the alternative macrophage markers C-C motif chemokine 18 (CCL18) and the hemoglobin scavenger receptor CD163 as well as platelet factor 4 (CXCL4) and platelet basic protein (CXCL7) with "platelet alpha granule" being returned as the top category according to the Gene Ontology Annotation. Apart from cathepsin L, the cathepsin L inhibitor also attenuated the release of a wide range of other cathepsins and lysosomal proteins such as legumain, but stimulated the secretion of members of the S100 protein family. The data presented here are the most comprehensive characterization of protein expression and secretion in human EPCs to date and highlight the potential importance of cysteine proteases in the processing of platelet factors for their pro-angiogenic potential. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited"

Effects of perhexiline-induced fuel switch on the cardiac proteome and metabolome.

Perhexiline is a potent anti-anginal drug used for treatment of refractory angina and other forms of heart disease. It provides an oxygen sparing effect in the myocardium by creating a switch from fatty acid to glucose metabolism through partial inhibition of carnitine palmitoyltransferase 1 and 2. However, the precise molecular mechanisms underlying the cardioprotective effects elicited by perhexiline are not fully understood. The present study employed a combined proteomics, metabolomics and computational approach to characterise changes in murine hearts upon treatment with perhexiline. According to results based on difference in-gel electrophoresis, the most profound change in the cardiac proteome related to the activation of the pyruvate dehydrogenase complex. Metabolomic analysis by high-resolution nuclear magnetic resonance spectroscopy showed lower levels of total creatine and taurine in hearts of perhexiline-treated mice. Creatine and taurine levels were also significantly correlated in a cross-correlation analysis of all metabolites. Computational modelling suggested that far from inducing a simple shift from fatty acid to glucose oxidation, perhexiline may cause complex rebalancing of carbon and nucleotide phosphate fluxes, fuelled by increased lactate and amino acid uptake, to increase metabolic flexibility and to maintain cardiac output. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism"

Effects of perhexiline-induced fuel switch on the cardiac proteome and metabolome

Perhexiline is a potent anti-anginal drug used for treatment of refractory angina and other forms of heart disease. It provides an oxygen sparing effect in the myocardium by creating a switch from fatty acid to glucose metabolism through partial inhibition of carnitine palmitoyltransferase 1 and 2. However, the precise molecular mechanisms underlying the cardioprotective effects elicited by perhexiline are not fully understood. The present study employed a combined proteomics, metabolomics and computational approach to characterise changes in murine hearts upon treatment with perhexiline. According to results based on difference in-gel electrophoresis, the most profound change in the cardiac proteome related to the activation of the pyruvate dehydrogenase complex. Metabolomic analysis by high-resolution nuclear magnetic resonance spectroscopy showed lower levels of total creatine and taurine in hearts of perhexiline-treated mice. Creatine and taurine levels were also significantly correlated in a cross-correlation analysis of all metabolites. Computational modelling suggested that far from inducing a simple shift from fatty acid to glucose oxidation, perhexiline may cause complex rebalancing of carbon and nucleotide phosphate fluxes, fuelled by increased lactate and amino acid uptake, to increase metabolic flexibility and to maintain cardiac output. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism"

Matrix metalloproteinase-8 promotes vascular smooth muscle cell proliferation and neointima formation

OBJECTIVE: We investigated the role of matrix metalloproteinase-8 (MMP8) in neointima formation and in vascular smooth muscle cell (VSMC) migration and proliferation.<p></p> APPROACH AND RESULTS: After carotid artery wire injuring, MMP8(-/-)/apoE(-/-) mice had fewer proliferating cells in neointimal lesions and smaller lesion sizes. Ex vivo assays comparing VSMCs isolated from MMP8 knockout and wild-type mice showed that MMP8 knockout decreased proliferation and migration. Proteomics analysis revealed that a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) had lower concentrations in MMP8 knockout VSMC culture media than in MMP8 wild-type VSMC culture media. Western blot, flow cytometric, and immunocytochemical analyses showed that MMP8 knockout VSMCs contained more pro-ADAM10 but less mature ADAM10, more N-cadherin, and β-catenin in the plasma membrane but less β-catenin in the nucleus and less cyclin D1. Treatment of MMP8 wild-type VSMCs with an ADAM10 inhibitor, GI254023X, or siRNA knockdown of ADAM10 in MMP8 wild-type VSMCs inhibited proliferation and migration, increased N-cadherin and β-catenin in the plasma membrane, reduced β-catenin in the nucleus, and decreased cyclin D1 expression. Incubation of MMP8 knockout VSMCs with a recombinant ADAM10 rescued the proliferative and migratory ability of MMP8 knockout VSMCs and increased cyclin D1 expression. Furthermore, immunohistochemical analyses showed colocalization of ADAM10 with VSMCs and N-cadherin, and nuclear accumulation of β-catenin in the neointima in apoE(-/-)/MMP8(+/+) mice.<p></p> CONCLUSIONS: MMP8 enhances VSMC proliferation via an ADAM10, N-cadherin, and β-catenin-mediated pathway and plays an important role in neointima formation.<p></p&gt