Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration
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
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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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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Figure 6

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Immunolocalization of MPO, NO2Tyr, and fibronectin in rat aortic rings. ...
Immunolocalization of MPO, NO2Tyr, and fibronectin in rat aortic rings. Rat aortic rings were exposed to MPO (65 nM) for 120 minutes, washed, and incubated with NO2– (100 μM) and H2O2 (50 μM) for 90 minutes. In some cases, vessel explants were preincubated with enoxaparin (150 μg·ml–1) and washed prior to MPO exposure and omitting the washing step before H2O2 and NO2– addition. Tissue antigen distribution was visualized using rabbit polyclonal anti-MPO (green), mouse monoclonal anti-NO2Tyr (red), and rabbit polyclonal anti-rat fibronectin (green). (a–c) Controls. Untreated vessel segments immunostained for MPO and NO2Tyr (a), vessel segments incubated with MPO and stained for NO2Tyr (b), and vessel segments treated with H2O2 and NO2 stained for NO2Tyr (c). (d–f) MPO and NO2Tyr distribution. Immunoreactivity for MPO (d) and NO2Tyr (e) colocalize, as shown when images were overlaid (f). (g–i) Effect of enoxaparin on NO2Tyr formation. Untreated vessel stained for NO2Tyr (g), MPO-catalyzed NO2Tyr formation (h) was reduced when vessel explants were preincubated with enoxaparin (i). (j–l) Vascular FN and NO2Tyr distribution. Subendothelial fibronectin immunoreactivity (j) colocalizes with NO2Tyr immunoreactivity (k), as shown in the merged image (l). L, vessel lumen; M, media. Arrows indicate endothelial cells. ×100 (a–f, j–l); ×50 (g–i).