title: Glyoxalase 1-knockdown in human aortic endothelial cells - effect on the proteome and endothelial function estimates.
abstract: Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage.
authors.affiliation: Herz und Diabeteszentrum NRW, Diabeteszentrum, Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Stratmann
authors.affiliation: Herz und Diabeteszentrum NRW, Diabeteszentrum, Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Engelbrecht
authors.affiliation: Herz und Diabeteszentrum NRW, Diabeteszentrum, Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Britta C
authors.initials: BC
authors.lastname: Espelage
authors.affiliation: Herz und Diabeteszentrum NRW, Diabeteszentrum, Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Klusmeier
authors.affiliation: Herz und Diabeteszentrum NRW, Diabeteszentrum, Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Janina
authors.initials: J
authors.lastname: Tiemann
authors.affiliation: Herz und Diabeteszentrum NRW, Diabeteszentrum, Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Gawlowski
authors.affiliation: Herz und Diabeteszentrum NRW, Diabeteszentrum, Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Mattern
authors.affiliation: Medical Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Eisenacher
authors.affiliation: Medical Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Helmut E
authors.initials: HE
authors.lastname: Meyer
authors.affiliation: Warwick Medical School, Clinical Sciences Research Laboratories, University of Warwick, University Hospital, Coventry UK.
authors.forename: Naila
authors.initials: N
authors.lastname: Rabbani
authors.affiliation: Warwick Medical School, Clinical Sciences Research Laboratories, University of Warwick, University Hospital, Coventry UK.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Thornalley
authors.affiliation: Herz und Diabeteszentrum NRW, Diabeteszentrum, Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Diethelm
authors.initials: D
authors.lastname: Tschoepe
authors.affiliation: Medical Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.|Molecular Proteomics Laboratory, BMFZ, Heinrich-Heine-University, Düsseldorf, Germany.
authors.forename: Gereon
authors.initials: G
authors.lastname: Poschmann
authors.affiliation: Medical Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.|Molecular Proteomics Laboratory, BMFZ, Heinrich-Heine-University, Düsseldorf, Germany.|Institute for Molecular Medicine, Heinrich-Heine-University, Düsseldorf, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Stühler
mesh_terms: D001011:Aorta / Q000473:pathology; D017209:Apoptosis; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000276:immunology / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D048909:Diabetes Complications / Q000378:metabolism*; D019332:Endothelin-1 / Q000276:immunology / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007382:Intermediate Filaments / Q000378:metabolism; D007791:Lactoylglutathione Lyase / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020543:Proteome; D011765:Pyruvaldehyde / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D019332:Endothelin-1; C573420:ICAM1 protein, human; D020543:Proteome; D034741:RNA, Small Interfering; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D011765:Pyruvaldehyde; D003094:Collagen; C000597196:GLO1 protein, human; D007791:Lactoylglutathione Lyase