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.

Figure 1

Degradation of [¹²⁵I]LDL by hMDMs and MPMs after modification by activated human monocytes. Isolated human monocytes (10⁶/mL) were incubated at 37°C in HBSS supplemented with DTPA (100 μM), [¹²⁵I]LDL (0.2 mg/mL), and NO₂^– (500 μM). Monocytes were activated with phorbol ester (200 nM) and maintained in suspensions by intermittent inversion (complete system) for 8 hours. Additions or deletions to the complete system were as indicated. Reactions were stopped by addition of BHT (40 μM) and catalase (300 nM); the cells were pelleted; 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. The final concentrations of additions to the complete system were catalase (300 nM) and 3-aminotriazole (1 mM). Data represent the mean ± SD of 3 separate experiments. *P < 0.001 for complete system vs. complete system – NO₂^–. Atz, 3-aminotriazole; Mono, human peripheral blood monocyte.

Figure 2

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).

Figure 3

NO₂^– concentration dependence of MPO-mediated modification of LDL and conversion into a high-uptake form for macrophages. [¹²⁵I]LDL (0.2 mg/mL) was incubated with isolated human MPO (30 nM), glucose (100 μM), glucose oxidase (20 ng/mL), and the indicated concentrations of NO₂^–, in the absence (filled circles) and presence (filled squares) of Cl^– (100 mM) in sodium phosphate buffer (50 mM, pH 7.0) supplemented with DTPA (100 μM) overnight at 37°C, as described in Methods. Uptake of the modified lipoproteins was then assessed using hMDMs (a) and thioglycollate-elicited MPMs (b) as described in Methods. Data represent the mean ± SD of triplicate determinations. Similar results were observed in 3 independent experiments.

Figure 4

LDL protein nitration, lipid peroxidation, and conversion into a high-uptake form for macrophages mediated by the MPO-H₂O₂-NO₂^– system. [¹²⁵I]LDL (0.2 mg/mL) was incubated with isolated human MPO (30 nM), glucose (100 μM), glucose oxidase (20 ng/mL), and NO₂^– (500 μM) in sodium phosphate buffer (50 mM, pH 7.0) supplemented with DTPA (100 μM) (complete system) or the indicated additions or deletions. The degradation of lipoproteins by thioglycollate-elicited MPMs (a), the content of nitrotyrosine (b), and TBA reactive products (c) generated were then determined as described in Methods. Data represent the mean ± SD for 3 independent experiments. *P < 0.001 for complete system vs. complete system – NO₂^–.

Figure 5

Time course of LDL conversion into a high-uptake form after exposure to the MPO-H₂O₂-NO₂^– system. [¹²⁵I]LDL (0.2 mg/mL) was incubated with isolated human MPO (30 nM), glucose (100 μM), and glucose oxidase (20 ng/mL) in sodium phosphate buffer (50 mM, pH 7.0) supplemented with DTPA (100 μM) for the indicated times in either the presence (+ NO₂^–; filled circles) or absence (– NO₂^–; open circles) of NO₂^– (500 μM) as described in Methods. Uptake of the modified lipoprotein by hMDMs (a) and thioglycollate-elicited MPMs (b) was then determined as described in Methods. Data represent the mean ± SD for triplicate determinations. Similar results were observed in 3 independent experiments.

Figure 6

Characterization of multiple protein components of LDL exposed to the MPO-H₂O₂-NO₂^– system. LDL samples prepared for Figure 5 were analyzed for nitrotyrosine content (a) tryptophan fluorescence (b), unmodified N^ε-lysine groups (c), and lipoprotein REM (d), as described in Methods. Under the conditions used, the REM for exhaustively acetylated LDL is 3.8. Data represent the mean ± SD of 3 independent experiments (1 using ¹²⁵I-labeled LDL as starting material, and 2 using nonlabeled LDL as starting material). + NO₂^– (filled circles), LDL modified by MPO, an H₂O₂-generating system (GGOx), and NO₂^– as described in Figure 5; – NO₂^– (open circles), LDL modified by MPO and an H₂O₂-generating system (GGOx) as described in Figure 5.

Figure 7

Quantification of multiple lipid oxidation products formed during LDL modification by the MPO-H₂O₂-NO₂^– system. LDL samples prepared for Figure 5 were analyzed for TBA reactive products (a), total lipid hydroperoxides (b), total 9-H(P)ODE (c), and cholesteryl-9-H(P)ODE (d) as described in Methods. Data represent the mean ± SD of 3 independent experiments (1 using [¹²⁵I]LDL as starting material and 2 using nonlabeled LDL as starting material). + NO₂^– (filled circles), LDL modified by MPO, an H₂O₂-generating system (GGOx), and NO₂^– as described in Figure 5; – NO₂^– (open circles), LDL modified by MPO and an H₂O₂-generating system (GGOx) as described in Figure 5.

Figure 8

(a) Size exclusion chromatography. (b) The effect of cytochalasin D treatment on macrophage degradation of modified forms of LDL. (a) Native [¹²⁵I]LDL (LDL; filled circles), copper oxidized [¹²⁵I]LDL (oxLDL; filled squares), and [¹²⁵I]LDL modified by the complete MPO-H₂O₂-NO₂^– system (NO₂-LDL; filled triangles) were prepared and individually fractionated on a Sephacryl S400-HR column as described in Methods. The elution profiles of native and modified lipoproteins are shown. The void volume (v) of the column is indicated. (b) Acetylated [¹²⁵I]LDL (acLDL), vortex-aggregated [¹²⁵I]LDL (aggrLDL), and [¹²⁵I]LDL modified by the complete MPO-H₂O₂-NO₂^– system (NO₂-LDL) were prepared as described in Methods. The ¹²⁵I-labeled lipoprotein preparations were then individually incubated (5 μg/mL) with thioglycollate-elicited MPMs at 37°C for 5 hours in the presence or absence of cytochalasin D (1 μg/mL) in media supplemented with catalase (300 nM) and BHT (20 μM). Cellular degradation of lipoprotein was subsequently determined as described in Methods. Results are expressed as the percentage of lipoprotein degradation observed in the presence vs. absence of cytochalasin D treatment. Lipoprotein degradation by non-cytochalasin D–treated macrophages (control) exposed to acLDL, aggrLDL, and NO₂-LDL preparations was 3.69 ± 0.14, 1.78 ± 0.03, and 1.49 ± 0.08 μg LDL per milligram of cell protein, respectively. Data represent the mean ± SD of triplicate determinations from a representative experiment performed in duplicate.

Figure 9

Concentration dependence of macrophage binding (a) and degradation (b) of LDL modified by the MPO-H₂O₂-NO₂^– system. (a) [¹²⁵I]LDL was modified in the presence of MPO and an H₂O₂-generating system (GGOx) in either the presence (+ NO₂^–; filled circles) or absence (– NO₂^–; open circles) of NO₂^– in sodium phosphate buffer (50 mM, pH 7.0) supplemented with DTPA (100 μM) as described in Methods. Thioglycollate-elicited MPM binding (a) and degradation (b) of the indicated concentrations of lipoproteins were then assessed as described in Methods. Data represent the mean of duplicate determinations (binding studies) or the mean ± SD of triplicate determinations (degradation studies). Similar results were observed in 3 independent experiments.

Figure 10

Uptake of ¹²⁵I-labeled lipoproteins by CHO cells transfected with the murine scavenger receptor class A type I. Confluent CHO cells expressing the murine scavenger receptor class A type I (white bars) or control vector-transfected parental CHO cells that lack the LDL receptor (black bars) were incubated for 5 hours at 37°C with 5 μg/mL of the indicated ¹²⁵I-labeled lipoproteins prepared as described in Methods. Cellular uptake of modified lipoproteins was then determined as described in Methods. Data represent the mean ± SD of triplicate determinations. Similar results were observed in 3 independent experiments. *P < 0.05 vs. control vector-transfected parental CHO cells; **P < 0.001 vs. control vector-transfected parental CHO cells. acLDL, acetylated LDL; LDL, native LDL; mSR-AI, CHO cells expressing the murine scavenger receptor class A type I; + NO₂-LDL, LDL modified by the complete MPO-H₂O₂-NO₂^- system; – NO₂-LDL, LDL modified by the complete system in the absence of NO₂^–; oxLDL, copper oxidized (5 hours) LDL.

Figure 11

Cholesteryl oleate synthesis (a), cholesteryl ester mass (b), and cholesterol mass (c) of cells exposed to LDL modified by the MPO-H₂O₂-NO₂^– system. [¹²⁵I]LDL (0.2 mg/mL) was incubated with isolated human MPO (30 nM), glucose (100 μM), glucose oxidase (20 ng/mL), and NO₂^– (500 μM) in sodium phosphate buffer (50 mM, pH 7.0) supplemented with DTPA (100 μM) (complete system) or the indicated additions or deletions. Lipoproteins were then incubated with thioglycollate-elicited MPMs, and the extent of cholesteryl [¹⁴C]oleate formation (a), cellular cholesteryl ester mass (b), and free cholesterol mass (c) was determined as described in Methods. Data represent the mean ± SD of triplicate determinations. Similar results were observed in 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 for comparison vs. LDL modified by MPO and an H₂O₂-generating system only (– NO₂^–). No significant cholesteryl [¹⁴C]oleate formation or increase in cholesteryl ester or free cholesterol mass above basal values was observed in LDL preparations modified by the complete system in the absence of either MPO or GGOx. Hi catalase, heat-inactivated catalase.

Figure 12

Macrophage foam cell formation by exposure to LDL modified by the MPO-H₂O₂-NO₂^– system. Thioglycollate-elicited MPMs were grown for 48 hours in RPMI-1640 containing 10% FBS. Cells were then incubated for 72 hours in the same media containing catalase (100 nM), BHT (20 μM), and vitamin E (20 μM) in the presence of the following lipoprotein preparations (50 μg/mL): LDL (0.2 mg/mL) previously incubated with isolated human MPO (30 nM), glucose (100 μM), and glucose oxidase (20 ng/mL) in sodium phosphate buffer (50 mM, pH 7.0) supplemented with DTPA (100 μM) at 37°C for 8 hours in the presence (+ NO₂^–) and absence (– NO₂^–) of NO₂^– (500 μM), native LDL (LDL), and acetylated LDL (acLDL). Media containing the appropriate modified lipoproteins were exchanged after 24-hour incubation. Cells were fixed with 4% formaldehyde and stained with hematoxylin and oil red O. Original magnification: ×300.

Figure 13

High-power magnification view of macrophage foam cells formed by exposure to LDL modified by the MPO-H₂O₂-NO₂^– system and acetylated LDL (acLDL). Differential interference contrast microscopy of hematoxylin and oil red O–stained thioglycollate-elicited MPMs exposed to NO₂-LDL and acLDL as in Figure 12. Scale bar: 10 μm.