Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration
Stephan Baldus, … , Kevin P. Moore, Bruce A. Freeman
Stephan Baldus, … , Kevin P. Moore, Bruce A. Freeman
Published December 15, 2001
Citation Information: J Clin Invest. 2001;108(12):1759-1770. https://doi.org/10.1172/JCI12617.
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Article

Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration

Abstract

Nitrotyrosine formation is a hallmark of vascular inflammation, with polymorphonuclear neutrophil–derived (PMN-derived) and monocyte-derived myeloperoxidase (MPO) being shown to catalyze this posttranslational protein modification via oxidation of nitrite (NO2–) to nitrogen dioxide (NO2•). Herein, we show that MPO concentrates in the subendothelial matrix of vascular tissues by a transcytotic mechanism and serves as a catalyst of ECM protein tyrosine nitration. Purified MPO and MPO released by intraluminal degranulation of activated human PMNs avidly bound to aortic endothelial cell glycosaminoglycans in both cell monolayer and isolated vessel models. Cell-bound MPO rapidly transcytosed intact endothelium and colocalized abluminally with the ECM protein fibronectin. In the presence of the substrates hydrogen peroxide (H2O2) and NO2–, cell and vessel wall–associated MPO catalyzed nitration of ECM protein tyrosine residues, with fibronectin identified as a major target protein. Both heparin and the low–molecular weight heparin enoxaparin significantly inhibited MPO binding and protein nitrotyrosine (NO2Tyr) formation in both cultured endothelial cells and rat aortic tissues. MPO–/– mice treated with intraperitoneal zymosan had lower hepatic NO2Tyr/tyrosine ratios than did zymosan-treated wild-type mice. These data indicate that MPO significantly contributes to NO2Tyr formation in vivo. Moreover, transcytosis of MPO, occurring independently of leukocyte emigration, confers specificity to nitration of vascular matrix proteins.

Authors

Stephan Baldus, Jason P. Eiserich, Alireza Mani, Laura Castro, Mario Figueroa, Phillip Chumley, Wenxin Ma, Albert Tousson, C. Roger White, Daniel C. Bullard, Marie-Luise Brennan, Aldons J. Lusis, Kevin P. Moore, Bruce A. Freeman

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MPO-dependent tyrosine nitration of ECM fibronectin. (a) NO2Tyr formatio...
MPO-dependent tyrosine nitration of ECM fibronectin. (a) NO2Tyr formation in cultured endothelial cell ECM proteins. Confluent BAEC monolayers were exposed to MPO (13 nM), and washed prior to NO2– (100 μM) and H2O2 (50 μM) addition. In some cases, cells were exposed to enoxaparin (Enox, 150 μg·ml–1) and washed, followed by MPO exposure and no washing, before NO2– and H2O2 were added. Matrix-enriched protein fractions were isolated as described in Methods, separated by 4–20% SDS-PAGE gradient gels, and probed with mouse monoclonal anti-fibronectin and rabbit anti-NO2Tyr. Protein staining with Coomassie blue confirmed that equal amounts of protein were electrophoretically resolved in each exposure condition. (b–d) NO2Tyr formation in purified human fibronectin and its major fragments. Fibronectin (100 μg·ml–1) (b) and the 30-, 45-, and 70-kDa fragments of fibronectin (d) were incubated with MPO (26 nM), μM NO2– (20–80 μM), and H2O2 (50 μM) in HBSS for 90 minutes. In some cases, fibronectin (c) and fibronectin fragments (d) were preincubated with enoxaparin (15 and 150 μg·ml–1) for 45 minutes before MPO, NO2–, and H2O2 addition. Proteins were separated by SDS-PAGE electrophoresis (7.5% gels for fibronectin and 10% gels for fibronectin fragments) and then immunoblotted with rabbit polyclonal anti-NO2Tyr.