Myeloperoxidase-generated reactive nitrogen species convert LDL into an atherogenic form in vitro
Eugene A. Podrez, … , Henry F. Hoff, Stanley L. Hazen
Eugene A. Podrez, … , Henry F. Hoff, Stanley L. Hazen
Published June 1, 1999
Citation Information: J Clin Invest. 1999;103(11):1547-1560. https://doi.org/10.1172/JCI5549.
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Article

Myeloperoxidase-generated reactive nitrogen species convert LDL into an atherogenic form in vitro

Abstract

Oxidized LDL is implicated in atherosclerosis; however, the pathways that convert LDL into an atherogenic form in vivo are not established. Production of reactive nitrogen species may be one important pathway, since LDL recovered from human atherosclerotic aorta is enriched in nitrotyrosine. We now report that reactive nitrogen species generated by the MPO-H2O2-NO2– system of monocytes convert LDL into a form (NO2-LDL) that is avidly taken up and degraded by macrophages, leading to massive cholesterol deposition and foam cell formation, essential steps in lesion development. Incubation of LDL with isolated MPO, an H2O2-generating system, and nitrite (NO2–)— a major end-product of NO metabolism—resulted in nitration of apolipoprotein B 100 tyrosyl residues and initiation of LDL lipid peroxidation. The time course of LDL protein nitration and lipid peroxidation paralleled the acquisition of high-affinity, concentration-dependent, and saturable binding of NO2-LDL to human monocyte–derived macrophages and mouse peritoneal macrophages. LDL modification and conversion into a high-uptake form occurred in the absence of free metal ions, required NO2–, occurred at physiological levels of Cl–, and was inhibited by heme poisons, catalase, and BHT. Macrophage binding of NO2-LDL was specific and mediated by neither the LDL receptor nor the scavenger receptor class A type I. Exposure of macrophages to NO2-LDL promoted cholesteryl ester synthesis, intracellular cholesterol and cholesteryl ester accumulation, and foam cell formation. Collectively, these results identify MPO-generated reactive nitrogen species as a physiologically plausible pathway for converting LDL into an atherogenic form.

Authors

Eugene A. Podrez, David Schmitt, Henry F. Hoff, Stanley L. Hazen

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Degradation of [125I]LDL by hMDMs and MPMs after modification by MPO-gen...
Degradation of [125I]LDL by hMDMs and MPMs after modification by MPO-generated chlorinating and nitrating intermediates. [125I]LDL (0.2 mg/mL) was incubated with isolated human MPO (30 nM), glucose (100 μM), and glucose oxidase (20 ng/mL) in the presence of the indicated additions in sodium phosphate buffer (50 mM, pH 7.0) supplemented with DTPA (100 μM) overnight at 37°C, as described in Methods. Under these conditions, a constant flux of H2O2 (0.18 μM/min) is generated by the GGOx system. Reactions were stopped by addition of BHT (40 μM) and catalase (300 nM), and then 125I-labeled lipoproteins (5 μg/mL) were incubated with either hMDMs (a) or thioglycollate-elicited MPMs (b) at 37°C for 5 hours in the appropriate media containing additional catalase (300 nM) and BHT (20 μM). Cellular uptake of lipoproteins was subsequently determined as described in Methods. When indicated, Cl– (100 mM) or NO2– (500 μM) were added during LDL modification by MPO. Data represent the mean ± SD of triplicate determinations. Similar results were observed in 3 independent experiments. *P < 0.001 for comparison vs. LDL modified in the presence of MPO and an H2O2-generating system (LDL/MPO/GGOx).