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. 2009 Nov 6;284(45):30825-35.
doi: 10.1074/jbc.M109.047605. Epub 2009 Sep 2.

Modification of high density lipoprotein by myeloperoxidase generates a pro-inflammatory particle

Arundhati Undurti  1 , Ying Huang, Joseph A Lupica, Jonathan D Smith, Joseph A DiDonato, Stanley L Hazen
Affiliations

Modification of high density lipoprotein by myeloperoxidase generates a pro-inflammatory particle

Arundhati Undurti et al. J Biol Chem. .

Abstract

High density lipoprotein (HDL) is the major atheroprotective particle in plasma. Recent studies demonstrate that myeloperoxidase (MPO) binds to HDL in vivo, selectively targeting apolipoprotein A1 (apoA1) of HDL for oxidative modification and concurrent loss in cholesterol efflux and lecithin cholesterol acyl transferase activating activities, generating a "dysfunctional HDL" particle. We now show that (patho)physiologically relevant levels of MPO-catalyzed oxidation result in loss of non-cholesterol efflux activities of HDL including anti-apoptotic and anti-inflammatory functions. One mechanism responsible is shown to involve the loss of modified HDL binding to the HDL receptor, scavenger receptor B1, and concurrent acquisition of saturable and specific binding to a novel unknown receptor independent of scavenger receptors CD36 and SR-A1. HDL modification by MPO is further shown to confer pro-inflammatory gain of function activities as monitored by NF-kappaB activation and surface vascular cell adhesion molecule levels on aortic endothelial cells exposed to MPO-oxidized HDL. The loss of non-cholesterol efflux activities and the gain of pro-inflammatory functions requires modification of the entire particle and can be recapitulated by oxidation of reconstituted HDL particles comprised of apoA1 and nonoxidizable phosphatidylcholine species. Multiple site-directed mutagenesis studies of apoA1 suggest that the pro-inflammatory activity of MPO-modified HDL does not involve methionine, tyrosine, or tryptophan, oxidant-sensitive residues previously mapped as sites of apoA1 oxidation within human atheroma. Thus, MPO-catalyzed oxidation of HDL results not only in the loss of classic atheroprotective reverse cholesterol transport activities of the lipoprotein but also both the loss of non-cholesterol efflux related activities and the gain of pro-inflammatory functions.

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Figures

FIGURE 1.
FIGURE 1.
Oxidation of HDL by the MPO/H2O2/Cl− system has functional consequences for classic atheroprotective activities of HDL. A, RAW macrophages were loaded with [3H]cholesterol and incubated for 6 h with 100 μg of protein/ml of HDL, HDL oxidized by the complete MPO/H2O2/Cl− system (oxHDL), or HDL exposed to the indicated components of the complete MPO system. The percentage cholesterol efflux was determined as described under “Experimental Procedures.” B, demonstration that MPO-catalyzed oxidation of reconstituted nascent HDL inhibits LCAT activating activity. C, the content of protein bound chlorotyrosine on HDL exposed to the MPO/H2O2/Cl− system (or the indicated components) was determined by stable isotope dilution liquid chromatography-tandem mass spectrometry as described under “Experimental Procedures.” The arrow indicates the upper range of chlorotyrosine content reported in apoA1 recovered from human atherosclerotic plaque (15). The results represent the means of triplicate determinations of a representative experiment performed at least three times.
FIGURE 2.
FIGURE 2.
HDL protects HUVEC and BAEC from multiple apoptogenic triggers, whereas MPO-oxidized HDL fails to do so. A, HUVEC were placed in serum-free medium along with the indicated treatments for 6 h. B, apoptosis was quantified by either annexin positive staining by flow cytometry or TUNEL staining. C and D, HUVEC and BAEC were exposed to 254-nm UV irradiation for 10 min followed by incubation with the indicated treatments for 6 h. Apoptosis was quantified by TUNEL staining. The results represent the means of triplicate determinations of a representative experiment performed at least three times.
FIGURE 3.
FIGURE 3.
Exposure of HDL to the MPO oxidant system inhibits the anti-apoptotic activity of the particle as monitored by loss of capacity to both inhibit caspase-3 activity and induce eNOS activity. A, serum starvation of HUVEC for 24 h increases endothelial cell caspase-3 activity. The anti-apoptotic activity of HDL as monitored by inhibition in caspase-3 activation. The anti-apoptotic activity of HDL exposed to the complete MPO/H2O2/Cl− system is also shown. B, the capacity of HDL or HDL previously exposed to the MPO/H2O2/Cl− system to activate endothelial cell eNOS was determined by monitoring [3H]citrulline formation from [3H]arginine as described under “Experimental Procedures.” The results represent the means of triplicate determinations of a representative experiment performed at least three times.
FIGURE 4.
FIGURE 4.
HDL oxidized by physiologically relevant levels MPO-generated oxidants inhibits the anti-inflammatory activity of the particle in HUVEC and promotes VCAM-1 protein expression in BAEC independent of TNF-α. A, HUVEC surface VCAM-1 protein expression was quantified by enzyme-linked immunosorbent assay in the absence and presence of TNFα as described under “Experimental Procedures.” In parallel, the impact of concomitant incubation with 500 μg of protein/ml of HDL, HDL previously exposed to the complete MPO/H2O2/Cl− system (oxHDL), or HDL incubated with the indicated components of the MPO/H2O2/Cl− system, on HUVEC surface VCAM-1 protein levels was determined by enzyme-linked immunosorbent assay as described under “Experimental Procedures.” *, p < 0.05. B, BAEC were incubated with the indicated concentrations of isolated human HDL or HDL exposed to the complete MPO/H2O2/Cl− system (oxHDL), and VCAM-1 surface protein levels were determined as described under “Experimental Procedures.” C, BAEC were incubated with 500 μg of protein/ml of HDL previously exposed to the indicated components of the MPO oxidation system and surface VCAM-1 protein levels were determined by enzyme-linked immunosorbent assay as described under “Experimental Procedures.” D, BAEC were incubated for 6 h with 500 μg of protein/ml of HDL, oxHDL, isolated human apoA1, apoA1 previously exposed to the complete MPO/H2O2/Cl− system (oxApoA1), lipid extract of HDL, small unilamellar vesicles (SUV) comprised of POPC, lipid extract of oxHDL, POPC small unilamellar vesicles exposed to the complete MPO/H2O2/Cl− system (oxPOPC), reconstituted nascent HDL (rHDL), or rHDL exposed to the complete MPO/H2O2/Cl− system (ox rHDL), and then cell surface VCAM-1 protein levels in BAEC were determined as described under “Experimental Procedures.” NA represents “no addition.” All of the results represent the means of triplicate determinations of a representative experiment performed at least three times.
FIGURE 5.
FIGURE 5.
MPO-oxidized HDL induces bovine aortic endothelial cell NF-κB activation, IKK activation, and phosphorylation of IκBα. A, BAEC were incubated with TNFα for 30 min (first through third lanes), media only (NA), HDL for 3 h, or oxHDL for the indicated times. EMSA for NF-κB activation were then performed in whole cell lysates as described under “Experimental Procedures.” Where indicated, the lysates were also incubated with anti-NF-κB p65 or isotype control IgG and supershift (SS) of the NF-κB complex monitored. Parallel immunoblots (IB) were generated using phosphoserine 32- and 36-specific IκB-α antibody (p-IκB-α), demonstrating phosphorylation of IκB-α on serines 32 and 36 in oxHDL-treated cells. Immunoblot with specific antibody to IκB-α is also shown, along with a immunoblot of lysates probed with anti-β-actin to demonstrate equal loading in each lane. B, EMSA analysis of BAEC lysates as in A except that the cells were exposed to HDL modified by the complete MPO/H2O2/Cl− system (oxHDL) or the complete oxidant system minus the indicated components (i.e. −MPO, −H2O2, or −Cl−). Note that BAEC NF-κB activation is only observed by exposure to HDL previously incubated with the complete MPO/H2O2/Cl− system because eliminating any one of the components of the oxidation system produces a HDL particle that fails to activate endothelial cell NF-κB. C, BAEC were incubated with TNFα (30 min) as positive control, media alone (NA) as negative control, or either HDL (3 h) or HDL previously exposed to the complete MPO/H2O2/Cl− system (oxHDL, 2 or 3 h). IKK activity was then determined in BAEC lysates using IKK-specific immuno-pulldown coupled kinase assay (KA). IKK complex was immunoprecipitated with antibody to IKKγ, and kinase activity using recombinant GST-IκBα(1–54) and [32P]ATP as substrate was performed as described under “Experimental Procedures.” Note that IκBα is phosphorylated in response to stimulation by TNFα and oxHDL but not HDL. Specificity of the kinase reaction was confirmed by demonstrating failure of the site-specific mutant GST-IκB-α(1–54) (32A/36A) to be phosphorylated in TNFα-stimulated extracts. Parallel immunoblots using antibodies specific to either IKKγ or β-actin are also shown. Equivalent levels of GST-IκB-α substrate addition to the IKK complexes are shown by Coomassie Blue (CB) staining. NA refers to “no addition.”
FIGURE 6.
FIGURE 6.
MPO-oxidized HDL fails to bind to the physiologic HDL receptor SR-B1 and gains binding to an alternate receptor on endothelial cells. Top, specific binding of HDL and HDL previously oxidized by exposure to the complete MPO/H2O2/Cl− system (oxHDL) were determined on 293T human embryonic kidney cells transiently transfected with either human SR-B1 or vector as described under “Experimental Procedures.” Bottom, specific binding of HDL previously modified by the complete MPO/H2O2/Cl− system determined using BAECs as described under “Experimental Procedures.” The results represent the means of triplicate determinations of a representative experiment performed at least three times.
FIGURE 7.
FIGURE 7.
The scavenger receptors CD36 and SR-A1 do not recognize HDL modified by the MPO/H2O2/Cl− system. Specific binding of HDL previously modified by the complete MPO/H2O2/Cl− system (oxHDL) to the indicated MPMs was determined as described under “Experimental Procedures.” Note that oxHDL binds equally well to wild type MPMs (A) and DKO MPMs (B). The results represent the means of triplicate determinations of a representative experiment performed at least three times.
FIGURE 8.
FIGURE 8.
ApoA1 tyrosine, tryptophan, and methionine residues do not appear to be involved in endothelial activation by MPO-oxidized HDL. A, dose-response curve of isolated human apoA1-mediated cholesterol efflux activity (ABCA1-dependent) from cholesterol-laden RAW macrophages. B, ABCA1-mediated cholesterol efflux activity of various apoA1 in the absence versus presence of MPO-catalyzed oxidation was examined in RAW macrophages at subsaturating levels of protein (5 μg/ml). ApoA1 forms used included isolated human apoA1 (h-ApoA1), recombinant human apoA1 (rh-ApoA1), and the indicated site-directed mutant forms of recombinant human apoA1. 4WF represents recombinant human apoA1 in which the endogenous tryptophans at residues 8, 50, 72, and 108 were converted to phenylalanine. 3MV represents recombinant human apoA1 in which endogenous methionines at residues 86, 112, and 148 were converted to valine. 7YF represents recombinant human apoA1 in which endogenous tyrosines at residues 18, 29, 100, 115, 166, 192, and 236 were converted to phenylalanine. Note that oxidation by the complete MPO system substantially inhibits ABCA1-mediated cholesterol efflux from all apoA1 forms examined except for the oxidant-resistant 4WF mutant. C, recombinant HDL (rHDL) were generated using each of the recombinant human apoA1 forms indicated in B. The capacity of the indicated rHDL to promote BAEC activation in native form versus following oxidation by the MPO/H2O2/Cl− system was then determined by quantifying endothelial cell VCAM-1 surface protein levels. NA refers to “no addition.” Wt refers to rHDL generated with the wild type human sequence for apoA1. D, competition binding data demonstrating that excess MPO-oxidized rHDL generated with the 4WF apoA1 mutant significantly inhibits binding of oxHDL to BAECs. The results represent the means of triplicate determinations of a representative experiment performed at least three times.
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