Endothelial cell (EC) glycocalyx (GLX) comprise a multicomponent layer of proteoglycans and glycoproteins. Alteration of its integrity contributes to chronic vascular inflammation and leads to the development of cardiovascular diseases. Myeloperoxidase (MPO), a highly abundant enzyme released by polymorphonuclear neutrophils, binds to the GLX and deleteriously affects vascular EC functions. The focus of this study was to elucidate the mechanisms of MPO-mediated alteration of GLX molecules, and to unravel subsequent changes in endothelial integrity and function. MPO binding to GLX of human ECs and subsequent internalization was mediated by cell surface heparan sulfate chains. Moreover, interaction of MPO, which is carrying a cationic charge, with anionic glycosaminoglycans (GAGs) resulted in reduction of their relative charge. By means of micro-viscometry and atomic force microscopy, we disclosed that MPO can crosslink GAG chains. MPO-dependent modulation of GLX structure was further supported by alteration of wheat germ agglutinin staining. Increased expression of ICAM-1 documented endothelial cell activation by both catalytically active and also inactive MPO. Furthermore, MPO increased vascular permeability connected with reorganization of intracellular junctions, however, this was dependent on MPO's catalytic activity. Novel proteins interacting with MPO during transcytosis were identified by proteomic analysis. Altogether, these findings provide evidence that MPO through interaction with GAGs modulates overall charge of the GLX, causing modification of its structure and thus affecting EC function. Importantly, our results also suggest a number of proteins interacting with MPO that possess a variety of cellular localizations and functions.

Central European Institute for Technology Masaryk University Kamenice 5 Brno Czech Republic

Clinic of General and Interventional Cardiology Angiology Agnes Wittenborg Institute of Translational Cardiovascular Research Herz und Diabeteszentrum NRW Ruhr Universität Bochum Bad Oeynhausen Germany

Department of Biophysics of Immune System Institute of Biophysics of the Czech Academy of Sciences Kralovopolska 135 Brno Czech Republic

Faculty of Medicine Department of Histology and Embryology Masaryk University Kamenice 3 625 00 Brno Czech Republic

Institute of Biostatistics and Analyses Masaryk University Kamenice 3 Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 62500 Brno Czech Republic

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