This Article Abstract Full Text (PDF) Data Supplement Correction (v25,p264) All Versions of this Article: 24/12/2302    most recent 01.ATV.0000148703.43429.25v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marsche, G. Articles by Malle, E. Search for Related Content PubMed PubMed Citation Articles by Marsche, G. Articles by Malle, E. Related Collections Lipids Pathophysiology Other arteriosclerosis Lipid and lipoprotein metabolism Oxidant stress

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:2302.)
© 2004 American Heart Association, Inc.

Vascular Biology

2-Chlorohexadecanal Derived From Hypochlorite-Modified High-Density Lipoprotein–Associated Plasmalogen Is a Natural Inhibitor of Endothelial Nitric Oxide Biosynthesis

Gunther Marsche; Regine Heller; Günter Fauler; Alenka Kovacevic; Alexander Nuszkowski; Wolfgang Graier; Wolfgang Sattler; Ernst Malle

From the Institute of Molecular Biology and Biochemistry (G.M., A.K., W.G., W.S., E.M.) and the Clinical Institute of Medical and Chemical Laboratory Diagnostics (G.F.), Medical University Graz, Austria; and the Institute of Molecular Cell Biology (R.H., A.N.), University of Jena, Erfurt, Germany.

Correspondence to Dr Ernst Malle, Medical University Graz, Institute of Molecular Biology and Biochemistry, A-8010 Graz, Austria. E-mail ernst.malle{at}meduni-graz.at

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Objective— Myeloperoxidase, a heme enzyme that is present and active in human atherosclerotic lesions, provides a source for the generation of proinflammatory chlorinated reactants contributing to endothelial dysfunction. Modification of high-density lipoprotein (HDL) by hypochlorous acid/hypochlorite (HOCl/Oce^–)—generated in vivo by the myeloperoxidase-hydrogen peroxide-chloride system of activated phagocytes—forms a proatherogenic lipoprotein particle that binds to and is internalized by endothelial cells.

Methods and Results— Here we show that HDL, modified with physiologically relevant HOCl concentrations, attenuates the expression and activity of vasculoprotective endothelial nitric oxide synthase. HOCl-HDL promotes dislocalization of endothelial nitric oxide synthase from the plasma membrane and perinuclear location of human umbilical venous endothelial cells. We could identify 2-chlorohexadecanal as the active component mediating this inhibitory activity. This chlorinated fatty aldehyde is formed during HOCl-mediated oxidative cleavage of HDL-associated plasmalogen.

Conclusion— 2-Chlorohexadecanal, produced by the myeloperoxidase-hydrogen peroxide-chloride system of activated phagocytes may act as a mediator of vascular injury associated with ischemia-reperfusion injury, glomerulosclerosis, and atherosclerosis.

Modification of HDL by HOCl (generated in vivo by the myeloperoxidase–H₂O₂ system) promotes dislocalization of eNOS from the plasma membrane and perinuclear location of endothelial cells, and attenuates eNOS expression and NO biosynthesis. The component mediating this proatherogenic effect was identified as 2-chlorohexadecanal formed during HOCl-mediated attack of lipoprotein-associated plasmalogen.

Key Words: myeloperoxidase • 2-chlorinated fatty aldehyde • atherosclerosis • modified lipids • glomerulosclerosis • neutrophils

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Animal experimentation and clinical studies have provided convincing evidence that the known risk factors for cardiovascular disease can elicit a localized inflammatory response in the vasculature. The changes are most pronounced in endothelial cells and include oxidative stress, increased activation of endothelial signaling pathways, and the consequent adhesion, activation, and degranulation of leukocytes. The myeloperoxidase (MPO)-hydrogen peroxide-system of stimulated leukocytes, primarily neutrophils, generates hypochlorous acid/hypochlorite (HOCl/Oce^–), a potent bacterial oxidant in vivo.¹ MPO and HOCl are emerging as critical modulators of vascular injury by promoting inflammatory arterial pathology and subsequent formation of mature plaques. MPO is present and active in human lesion material.² HOCl reacts with a wide range of biological substrates, including antioxidants, amines, sulfides, nucleotides, DNA, lipids, and (lipo)proteins.^3–5 HOCl-modified (lipo)proteins are present in human^6–8 and rabbit lesions,^9,10 and disease stage-dependent accumulation of HOCl-modified (lipo)proteins has been reported.⁷

Elevated levels of plasma high-density lipoprotein (HDL) protect against atherosclerotic vascular disease. A broad spectrum of potent antioxidant and anti-inflammatory activities has been ascribed to native HDL.^11,12 However, oxidation/modification by HOCl alters the physicochemical and metabolic properties of anti-atherogenic HDL.^13–15 In vivo, apolipoprotein A-I, the major apolipoprotein of HDL, represents a selective target for MPO-catalyzed oxidation; a process apparently facilitated by MPO binding to apolipoprotein A-I.¹⁶ Furthermore, significantly increased levels of 3-chlorotyrosine, a protein modification specific for the MPO-hydrogen peroxide-chloride-catalyzed oxidation,¹⁷ were detected in association with apolipoprotein A-I recovered from serum of patients with cardiovascular disease.¹⁶ In situ experiments revealed colocalization of specific epitopes derived from HOCl with apolipoprotein A-I in human atherosclerotic lesions and on endothelial cells lining the blood vessel.⁸ MPO levels serve as a strong predictor of endothelial dysfunction in humans,¹⁸ and MPO colocalizes with HOCl-modified epitopes/(lipo)proteins on endothelial cells.

Native and modified lipoproteins have been reported to affect levels of nitric oxide (NO),^19–24 an ubiquitous signaling molecule modulating a plethora of physiological responses including the propagation of a number of anti-atherogenic effects in the endothelium.²⁵ Here, we show that treatment of human umbilical venous endothelial cells (HUVECs) with HDL modified by HOCl impairs expression and activity of endothelial nitric oxide synthase (eNOS). The active component mediating this effect was found to be 2-chlorohexadecanal (2-ClHDA) formed via HOCl-mediated oxidative attack of HDL-associated plasmalogen.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
A detailed Materials and Methods section on lipoproteins,^8,13,26 preparation of lipid micro-emulsions and acid hydrolysis of lipoprotein-associated lipids,^26,27 synthesis, derivatization, and quantitation of 2-ClHDA by gas chromatography–mass spectrometry,^28–30 cell culture experiments,²⁴ neutrophil stimulation,^9,27 eNOS activation assays,^24,31 Northern blot analysis and mRNA stability,²⁴ Western blotting,²⁴ and immunofluorescence is available online at http://atvb.ahajournals.org.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The effect of HDL to modulate NO release from endothelial cells has been investigated by several groups.³² Here, we show that HDL modified with HOCl concentrations that are easily achieved under acute inflammation^13,33 caused a marked, sustained, concentration-dependent, and time-dependent decrease of NO production in stimulated HUVECs as measured by the conversion of L-arginine to L-citrulline (Figure 1A to 1C). To confirm the functional significance of these findings, we assessed the effects of HOCl-HDL on the production of cGMP, a second messenger formed via activation of soluble guanylate cyclase by NO. As expected, HOCl-HDL caused a marked reduction of cellular cGMP production (Figure 1A and 1B). Of note, HOCl-HDL did not affect endothelial cell viability or alter cell growth behavior as determined by trypan blue exclusion and incorporation of ¹⁴C-labeled amino acids. Furthermore, no evidence of endothelial cell apoptosis was seen under the experimental conditions described (data not shown).

View larger version (37K): [in this window] [in a new window]   Figure 1. Ionomycin-induced L-citrulline and concomitant cGMP formation of HUVECs. Cells were preincubated for 18 hours in the absence (control) or presence of HDL or HOCl-HDL (A) at the indicated oxidant:lipoprotein molar ratio (90 µg HDL–total cholesterol [TC]/mL) and (B) increasing HOCl-HDL concentrations (oxidant:lipoprotein molar ratio of 50:1). C, Preincubation of HUVECs with HDL or HOCl-HDL (oxidant:lipoprotein molar ratio of 50:1; 90 µg TC/mL) for indicated time periods and subsequent ionomycin-induced L-citrulline biosynthesis (mean±SE, n=4). For experimental details, see Materials and Methods.

The efficacy of NO biosynthesis critically depends on the correct compartmentalization of eNOS in specific intracellular membrane domains, ie, Golgi complex and plasmalemmal caveolae.^34,35 We previously reported that low-density lipoprotein (LDL) modified by reagent HOCl inhibits NO synthesis in HUVECs via an intracellular dislocalization of eNOS without altering eNOS protein expression and without altering myristoylation or palmitoylation of the enzyme.²⁴ Here, we show that coincubation of HUVECs with HOCl-HDL also leads to changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the perinuclear location (Figure 2A). But in contrast to HOCl-LDL, HOCl-HDL further decreased eNOS expression on mRNA and protein level (Figure 2B and 2C). HOCl-HDL also induced a marked downregulation of eNOS protein expression when cells were incubated in the presence of DRB, a transcription inhibitor (Figure 2D). When cells were incubated with cycloheximide, an inhibitor of protein synthesis, HOCl-HDL had no significant effect indicating that a post-translational destabilization of eNOS is not involved; thus, it is conceivable that downregulation of eNOS is a post-transcriptional event caused by decreased stability of eNOS mRNA. In line with these observations, NO biosynthesis in cell lysates in the presence of saturating concentrations of substrate and cofactors³¹ was also decreased (Figure 2D). Of note, HOCl-HDL-induced decrease in L-citrulline formation was much more pronounced in ionomycin-stimulated intact cells than in cell lysates, suggesting that the combination of decreased eNOS protein content and eNOS mislocalization elicits the maximal inhibitory effect.

View larger version (85K): [in this window] [in a new window]   Figure 2. Expression and distribution of eNOS. HUVECs were treated in the absence (control) or presence of HDL or HOCl-HDL (oxidant:lipoprotein molar ratio of 50:1; 90 µg TC/mL). A, Confocal image of eNOS distribution. B, eNOS steady-state mRNA levels. C, eNOS protein expression in the absence or presence of 5,6-dichlorobenzimidazole 1-ß-D-ribofuranoside (DRB) or cycloheximide (CHX). D, L-Citrulline formation in cell lysates (mean±SE, n=3). For experimental details, see Materials and Methods.

Apolipoprotein A-I represents a preferential target for HOCl attack.^13,16,36,37 Treatment of HDL by HOCl leads also to consumption of unsaturated fatty acids.¹³ Furthermore, the formation of lysophospholipids from unsaturated phosphatidylcholines after HOCl treatment has been reported.³⁸ To identify the constituent of HOCl-HDL that interferes with eNOS biosynthesis, HUVECs were treated either with the lipoprotein–lipid extract (a micro-emulsion of isolated lipids) or with the remaining apolipoprotein moiety. Lipids extracted from HOCl-HDL inhibited ionomycin-induced L-citrulline synthesis in intact cells (Figure 3A) and eNOS protein expression (Figure 3B) to a similar extent as observed with intact HOCl-HDL. The apolipoprotein fraction of HOCl-HDL showed no inhibitory activity (data not shown). To further identify the active lipid component of HOCl-HDL, HDL-associated lipids were separated and modified with HOCl; an equivalent amount of these products being present in 90 µg HDL-TC/mL was used to treat HUVECs. Among the modified lipid subclasses tested, only the HOCl-modified plasmalogen fraction suppressed eNOS protein levels (Figure 3C). To further strengthen the potential role of these ether phospholipids, total HDL lipids were depleted from the plasmalogen fraction by acidic hydrolysis.²⁷ In line with data shown in Figure 3C, total HDL lipids modified with HOCl suppressed eNOS protein levels, whereas the plasmalogen-depleted lipid fraction, after modification with HOCl, was without effect on eNOS levels (Figure 3D).

View larger version (35K): [in this window] [in a new window]   Figure 3. Identification of the active component of HOCl-HDL. A, HUVECs were preincubated for 18 hours in the absence (control) or presence of HOCl-HDL (oxidant:lipoprotein molar ratio of 50:1) or HOCl-HDL–derived lipids. L-Citrulline formation after ionomycin stimulation is given as percentage of control (mean±SE, n=3). Western blot analysis of eNOS after treatment of HUVECs with HOCl-HDL (B) or HOCl-HDL–derived lipids (C), HOCl-modified-free cholesterol (FC), HOCl-cholesterol-linoleate (CE), HOCl-phosphatidylcholine (PC), HOCl-phosphatidylethanolamine (PE), HOCl-plasmalogen (PL, Avanti Polar Lipids), or (D) HDL-derived lipids (lane 2), HOCl-modified lipids (lane 3), and HOCl-modified plasmalogen-depleted lipids (lane 4); lane 1 (no addition of lipids).

Plasmalogens, localized in the plasma membranes of mammalian cells and present in lipoproteins,³⁹ are considered to be antioxidants because the vinyl ether bond is susceptible to attack by oxidants.³⁰ Recent findings revealed that the vinyl ether bonds of plasmalogens are highly accessible molecular targets of the membrane-permeable, reactive chlorinating species generated by the MPO-hydrogen peroxide-chloride system; as a result, lysophosphatidylcholine and -chlorinated fatty aldehydes are formed.³⁰ Thukkani et al²⁹ have shown that 2-ClHDA is formed as the main -chloro fatty aldehyde from cell-associated plasmalogen. In another report, these authors demonstrated that 2-ClHDA is also generated when LDL was coincubated with stimulated monocytes.⁴⁰ Therefore, we first tested whether HOCl (added as reagent) targets HDL-associated plasmalogen, leading to 2-ClHDA formation. For external quantification, 2-ClHDA was synthesized and characterized by gas chromatography–mass spectrometry analysis. Analysis of PFB oximes from synthetic 2-ClHDA and 2-ClHDA isolated from lipid extracts from HOCl-HDL (Figure I, available online at http://atvb.ahajournals.org) showed identical results regarding retention time of 7.25 minutes and 7.15 minutes (an expected C16:0 isoform),²⁹ as well as full scan spectra (Figures II and III, available online at http://atvb.ahajournals.org). Prominent fragmentation peaks occurred at m/z 196 and m/z 288/290. Additional peaks from lipid extracts obtained from HOCl-HDL could be identified as dichlorinated hexadecanal (7.42 minutes), monochlorinated C18:1 fatty aldehyde (7.70 minutes), and a monochlorinated C18:0 fatty aldehyde (7.77 minutes). 2-ClHDA could not be detected in native HDL (Figure I) or in plasmalogen (data not shown). When plasmalogen-enriched HDL was subjected to HOCl-treatment, 2-ClHDA concentrations increased several-fold compared with HOCl-HDL (data not shown).

The plasmalogen content in HDL is 100 nmol/mg TC.⁴² After HOCl-modification of HDL (oxidant:lipoprotein molar ratio of 100:1) 80% of the total plasmalogen fraction contributes to 2-ClHDA formation (Figure 4A). HOCl-modification of HDL (oxidant:lipoprotein molar ratio of 25:1 and 100:1; 90 µg TC/mL) resulted in the generation of 4 to 6.5 µmol/L 2-ClHDA. These levels are close to the concentration range of 2-ClHDA reported to be present in atherosclerotic lesions (10 µmol/L), a concentration that is almost 1400 times that of normal aorta samples.⁴¹ The plasmalogen content in LDL was found to be 74 nmol/mg TC, respectively.⁴² HOCl treatment of LDL (oxidant:lipoprotein molar ratio of 400:1) also led to generation of 2-ClHDA (data not shown). However, only up to 22% of LDL-associated plasmalogen was consumed after HOCl treatment.

View larger version (72K): [in this window] [in a new window]   Figure 4. Effect of 2-ClHDA on eNOS expression and NO biosynthesis. A, Formation of 2-ClHDA (measured by gas chromatography–mass spectrometry analysis) after modification of HDL by HOCl added as reagent at indicated oxidant:lipoprotein molar ratio or generated by phorbol-12-myristate-13-acetate (PMA)-stimulated neutrophils in the absence or presence of sodium azide. Effect of 2-ClHDA on eNOS activity and expression. HUVECs were incubated for 18 hours in the absence (control) or presence of 2-ClHDA. Agonist-induced L-citrulline formation (B) and confocal image of eNOS distribution (C). Western blot analysis of eNOS expression (D) and Northern blot experiments showing eNOS mRNA stability in the presence of 5,6-dichlorobenzimidazole 1-ß-D-ribofuranoside (DRB) (mean±SE, n=3) (E). For experimental details, see Materials and Methods.

Second, we tested whether HOCl—generated by the MPO-hydrogen peroxide-chloride system—will target the plasmalogen moiety of HDL, leading to the generation of 2-ClHDA. The predominant in vivo sources for MPO released during the oxidative burst are neutrophils and monocytes, in which MPO comprises 5% and 1% in the total protein content, respectively. Here, we show that considerable amounts of 2-ClHDA were generated via the active enzymatic pathway of stimulated neutrophils coincubated with native HDL (Figure 4A). In line with studies performed on monocytes,⁴⁰ the presence of the MPO inhibitor sodium azide (Figure 4A) or the HOCl scavenger methionine (data not shown) prevented 2-ClHDA formation after phorbolester activation of neutrophils.

To clarify whether 2-ClHDA directly alters eNOS expression and NO biosynthesis, 2-ClHDA was used in cell culture experiments. We present 4 lines of evidence that plasmalogen-derived 2-ClHDA is the lipid component mediating the inhibitory effects of HOCl-HDL on eNOS protein expression and activity. First, 2-ClHDA strongly inhibited ionomycin-stimulated NO formation (Figure 4B). Importantly, when cells were stimulated with the physiological agonist thrombin, the inhibitory activity of 2-ClHDA was even more pronounced. Second, as determined by confocal microscopy, 2-ClHDA affected intracellular eNOS targeting already at 2 µmol/L (Figure 4C). Higher concentrations of 2-ClHDA (5 and 10 µmol/L) led to a complete translocation of eNOS from the plasma membrane and disintegration of the perinuclear location. Third, 2-ClHDA—at concentrations of 5 µmol/L and higher—attenuated eNOS protein expression (Figure 4D). Fourth, 2-ClHDA (10 µmol/L) decreased eNOS mRNA expression in the presence of DRB (Figure 4E), indicating that eNOS mRNA stability is impaired. Of importance, effects of 2-ClHDA on eNOS protein expression were observed at threshold concentrations of 5 µmol/L, whereas disruption of eNOS localization was already observed at 2 µmol/L. This explains why LDL after HOCl treatment had no effect on eNOS protein expression,²⁴ as 2-ClHDA concentrations generated were calculated to be lower under our experimental conditions.

MPO gene variations may be considered as determinants of the atherosclerotic progression.⁴³ Elevated levels of leukocyte and blood MPO are predictive risk factors for endothelial dysfunction¹⁸ and coronary artery disease in humans.⁴⁴ Moreover, products of MPO-mediated reactions are present in human atherosclerotic lesions^{6–8,16,17,41,45} and MPO has been identified as an HDL-associated protein within human atheroma.¹⁶ Thus, it is conceivable that the MPO-hydrogen peroxide-chloride system acts as a platform for the formation of inflammatory markers generated during the initiation and progression of cardiovascular disease. HOCl-HDL is bound and internalized by HUVECs.⁴⁶ Also MPO—released by intraluminal degranulation of activated phagocytes—binds to and transcytoses across the endothelium⁴⁷ and may directly modulate vascular inflammatory responses by regulating NO bioavailability.⁴⁸ We show that 2-ClHDA—generated by reagent HOCl and the MPO-hydrogen peroxide-chloride system of activated phagocytes—is a strong modulator of eNOS expression and activity. Normally, eNOS produces a tonic amount of NO, which is responsible for the homeostasis between the endothelium and surrounding tissues. Our data indicate that HOCl-mediated generation of chlorinated fatty aldehydes from lipoprotein-associated plasmalogens could represent a dominant pathway contributing to pathophysiological changes, ultimately leading to inflammatory responses, endothelial dysfunction, and tissue injury.

	Acknowledgments

 
The authors thank Dr M. Vadon for providing human plasma, Dr A. Unbehaun for help with Northern blot experiments, and C. Reinprecht and M. Sundl for technical assistance. This work was supported by grants from the Deütsche Forschungsgemeinschaft (HE 2304/1–2), Österreichische Nationalbank (9962), and Austrian Science Fund (P15404, P17013, SFB F007/716) to R.H, W.S., and E.M.

Received July 24, 2004; accepted October 7, 2004.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 
1. Hampton MB, Kettle AJ, Winterbourn CC. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood. 1998; 92: 3007–3017.[Free Full Text]

2. Daugherty A, Dunn JL, Rateri DL, Heinecke JW. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest. 1994; 94: 437–444.

3. Winterbourn CC. Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarity of the oxidant to hypochlorite. Biochim Biophys Acta. 1985; 840: 204–210.[Medline] [Order article via Infotrieve]

4. Prutz WA. Hypochlorous acid interactions with thiols, nucleotides, DNA, and other biological substrates. Arch Biochem Biophys. 1996; 332: 110–120.[CrossRef][Medline] [Order article via Infotrieve]

5. Hazell LJ, Stocker R. Oxidation of low-density lipoprotein with hypochlorite causes transformation of the lipoprotein into a high-uptake form for macrophages. Biochem J. 1993; 290: 165–172.

6. Hazell LJ, Arnold L, Flowers D, Waeg G, Malle E, Stocker R. Presence of hypochlorite-modified proteins in human atherosclerotic lesions. J Clin Invest. 1996; 97: 1535–1544.[Medline] [Order article via Infotrieve]

7. Malle E, Waeg G, Schreiber R, Grone E, Sattler W, Grone HJ. Immunohistochemical evidence for the myeloperoxidase/H₂O₂/halide system in human atherosclerotic lesions: colocalization of myeloperoxidase and hypochlorite-modified proteins. Eur J Biochem. 2000; 267: 4495–4503.[Medline] [Order article via Infotrieve]

8. Marsche G, Hammer A, Oskolkova O, Kozarsky KF, Sattler W, Malle E. Hypochlorite-modified high density lipoprotein, a high affinity ligand to scavenger receptor class B, type I, impairs high density lipoprotein-dependent selective lipid uptake and reverse cholesterol transport. J Biol Chem. 2002; 277: 32172–32179.[Abstract/Free Full Text]

9. Malle E, Wag G, Thiery J, Sattler W, Grone HJ. Hypochlorite-modified (lipo)proteins are present in rabbit lesions in response to dietary cholesterol. Biochem Biophys Res Commun. 2001; 289: 894–900.[CrossRef][Medline] [Order article via Infotrieve]

10. Brasen JH, Hakkinen T, Malle E, Beisiegel U, Yla-Herttuala S. Patterns of oxidized epitopes, but not NF-kappa B expression, change during atherogenesis in WHHL rabbits. Atherosclerosis. 2003; 166: 13–21.[CrossRef][Medline] [Order article via Infotrieve]

11. Shah PK, Kaul S, Nilsson J, Cercek B. Exploiting the vascular protective effects of high-density lipoprotein and its apolipoproteins: an idea whose time for testing is coming, part I. Circulation. 2001; 104: 2376–2383.[Free Full Text]

12. Calabresi L, Gomaraschi M, Franceschini G. Endothelial protection by high-density lipoproteins: from bench to bedside. Arterioscler Thromb Vasc Biol. 2003; 23: 1724–1731.[Abstract/Free Full Text]

13. Panzenboeck U, Raitmayer S, Reicher H, Lindner H, Glatter O, Malle E, Sattler W. Effects of reagent and enzymatically generated hypochlorite on physicochemical and metabolic properties of high density lipoproteins. J Biol Chem. 1997; 272: 29711–29720.[Abstract/Free Full Text]

14. Bergt C, Reicher H, Malle E, Sattler W. Hypochlorite modification of high density lipoprotein: effects on cholesterol efflux from J774 macrophages. FEBS Lett. 1999; 452: 295–300.[CrossRef][Medline] [Order article via Infotrieve]

15. Pussinen PJ, Metso J, Keva R, Hirschmugl B, Sattler W, Jauhiainen M, Malle E. Plasma phospholipid transfer protein-mediated reactions are impaired by hypochlorite-modification of high density lipoprotein. Int J Biochem Cell Biol. 2003; 35: 192–202.[CrossRef][Medline] [Order article via Infotrieve]

16. Zheng L, Nukuna B, Brennan ML, Sun M, Goormastic M, Settle M, Schmitt D, Fu X, Thomson L, Fox PL, Ischiropoulos H, Smith JD, Kinter M, Hazen SL. Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease. J Clin Invest. 2004; 114: 529–541.[CrossRef][Medline] [Order article via Infotrieve]

17. Hazen SL, Heinecke JW. 3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J Clin Invest. 1997; 99: 2075–2081.[Medline] [Order article via Infotrieve]

18. Vita JA, Brennan ML, Gokce N, Mann SA, Goormastic M, Shishehbor MH, Penn MS, Keaney JF Jr, Hazen SL. Serum myeloperoxidase levels independently predict endothelial dysfunction in humans. Circulation. 2004; 110: 1134–1139.[Abstract/Free Full Text]

19. Chin JH, Azhar S, Hoffman BB. Inactivation of endothelial derived relaxing factor by oxidized lipoproteins. J Clin Invest. 1992; 89: 10–18.

20. Blair A, Shaul PW, Yuhanna IS, Conrad PA, Smart EJ. Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation. J Biol Chem. 1999; 274: 32512–32519.[Abstract/Free Full Text]

21. Vergnani L, Hatrik S, Ricci F, Passaro A, Manzoli N, Zuliani G, Brovkovych V, Fellin R, Malinski T. Effect of native and oxidized low-density lipoprotein on endothelial nitric oxide and superoxide production : key role of L-arginine availability. Circulation. 2000; 101: 1261–1266.[Abstract/Free Full Text]

22. Cominacini L, Rigoni A, Pasini AF, Garbin U, Davoli A, Campagnola M, Pastorino AM, Lo Cascio V, Sawamura T. The binding of oxidized low density lipoprotein (ox-LDL) to ox-LDL receptor-1 reduces the intracellular concentration of nitric oxide in endothelial cells through an increased production of superoxide. J Biol Chem. 2001; 276: 13750–13755.[Abstract/Free Full Text]

23. Chavakis E, Dernbach E, Hermann C, Mondorf UF, Zeiher AM, Dimmeler S. Oxidized LDL inhibits vascular endothelial growth factor-induced endothelial cell migration by an inhibitory effect on the Akt/endothelial nitric oxide synthase pathway. Circulation. 2001; 103: 2102–2107.[Abstract/Free Full Text]

24. Nuszkowski A, Gräbner R, Marsche G, Unbehaun A, Malle E, Heller R. Hypochlorite-modified low density lipoprotein inhibits nitric oxide synthesis in endothelial cells via an intracellular dislocalization of endothelial nitric-oxide synthase. J Biol Chem. 2001; 276: 14212–14221.[Abstract/Free Full Text]

25. Radomski MW, Moncada S. Regulation of vascular homeostasis by nitric oxide. Thromb Haemost. 1993; 70: 36–41.[Medline] [Order article via Infotrieve]

26. Marsche G, Zimmermann R, Horiuchi S, Tandon NN, Sattler W, Malle E. Class B scavenger receptors CD36 and SR-BI are receptors for hypochlorite-modified low density lipoprotein. J Biol Chem. 2003; 278: 47562–47570.[Abstract/Free Full Text]

27. Pugh EL, Kates M, Hanahan DJ. Characterization of the alkyl ether species of phosphatidylcholine in bovine heart. J Lipid Res. 1977; 18: 710–716.[Abstract]

28. Bellesia F, Boni M, Ghelfi F, Grandi R, Pagnoni UM, Pinetti A. Acetal chlorination with MnO₂-trimethylchlorosilane. Tetrahedron. 1992; 48: 4579–4586.[CrossRef]

29. Thukkani AK, Hsu FF, Crowley JR, Wysolmerski RB, Albert CJ, Ford DA. Reactive chlorinating species produced during neutrophil activation target tissue plasmalogens: production of the chemoattractant, 2-chlorohexadecanal. J Biol Chem. 2002; 277: 3842–3849.[Abstract/Free Full Text]

30. Albert CJ, Crowley JR, Hsu FF, Thukkani AK, Ford DA. Reactive chlorinating species produced by myeloperoxidase target the vinyl ether bond of plasmalogens: identification of 2-chlorohexadecanal. J Biol Chem. 2001; 276: 23733–23741.[Abstract/Free Full Text]

31. Heller R, Münscher-Paulig F, Gräbner R, Till U. L-Ascorbic acid potentiates nitric oxide synthesis in endothelial cells. J Biol Chem. 1999; 274: 8254–8260.[Abstract/Free Full Text]

32. Shaul PW, Mineo C. HDL action on the vascular wall: is the answer NO ? J Clin Invest. 2004; 113: 509–513.[CrossRef][Medline] [Order article via Infotrieve]

33. Katrantzis M, Baker MS, Handley CJ, Lowther DA. The oxidant hypochlorite (OCl-), a product of the myeloperoxidase system, degrades articular cartilage proteoglycan aggregate. Free Radic Biol Med. 1991; 10: 101–109.[CrossRef][Medline] [Order article via Infotrieve]

34. Sessa WC, Garcia-Cardena G, Liu J, Keh A, Pollock JS, Bradley J, Thiru S, Braverman IM, Desai KM. The Golgi association of endothelial nitric oxide synthase is necessary for the efficient synthesis of nitric oxide. J Biol Chem. 1995; 270: 17641–17644.[Abstract/Free Full Text]

35. Shaul PW. Regulation of endothelial nitric oxide synthase: location, location, location. Annu Rev Physiol. 2002; 64: 749–774.[CrossRef][Medline] [Order article via Infotrieve]

36. Bergt C, Oettl K, Keller W, Andreae F, Leis HJ, Malle E, Sattler W. Reagent or myeloperoxidase-generated hypochlorite affects discrete regions in lipid-free and lipid-associated human apolipoprotein A-I. Biochem J. 2000; 346: 345–354.

37. Bergt C, Marsche G, Panzenboeck U, Heinecke JW, Malle E, Sattler W. Human neutrophils employ the myeloperoxidase/hydrogen peroxide/chloride system to oxidatively damage apolipoprotein A-I. Eur J Biochem. 2001; 268: 3523–3531.[Medline] [Order article via Infotrieve]

38. Arnhold J, Osipov AN, Spalteholz H, Panasenko OM, Schiller J. Formation of lysophospholipids from unsaturated phosphatidylcholines under the influence of hypochlorous acid. Biochim Biophys Acta. 2002; 1572: 91–100.[Medline] [Order article via Infotrieve]

39. Paltauf F. Ether lipids in biomembranes. Chem Phys Lipids. 1994; 74: 101–139.[CrossRef][Medline] [Order article via Infotrieve]

40. Thukkani AK, Albert CJ, Wildsmith KR, Messner MC, Martinson BD, Hsu FF, Ford DA. Myeloperoxidase-derived reactive chlorinating species from human monocyte target plasmalogens in low density lipoprotein. J Biol Chem. 2003; 278: 36365–36372.[Abstract/Free Full Text]

41. Thukkani AK, McHowat J, Hsu FF, Brennan ML, Hazen SL, Ford DA. Identification of -chloro fatty aldehydes and unsaturated lysophosphatidylcholine molecular species in human atherosclerotic lesions. Circulation. 2003; 108: 3128–3133.[Abstract/Free Full Text]

42. Bräutigam C, Engelmann B, Reiss D, Reinhardt U, Thiery J, Richter WO, Brosche T. Plasmalogen phospholipids in plasma lipoproteins of normolipidemic donors and patients with hypercholesterolemia treated by LDL apheresis. Atherosclerosis. 1996; 119: 77–88.[CrossRef][Medline] [Order article via Infotrieve]

43. Makela R, Karhunen PJ, Kunnas TA, Ilveskoski E, Kajander OA, Mikkelsson J, Perola M, Penttila A, Lehtimaki T. Myeloperoxidase gene variation as a determinant of atherosclerosis progression in the abdominal and thoracic aorta: an autopsy study. Lab Invest. 2003; 83: 919–925.[CrossRef][Medline] [Order article via Infotrieve]

44. Zhang R, Brennan ML, Fu X, Aviles RJ, Pearce GL, Penn MS, Topol EJ, Sprecher DL, Hazen SL. Association between myeloperoxidase levels and risk of coronary artery disease. JAMA. 2001; 286: 2136–2142.[Abstract/Free Full Text]

45. Fogelman AM. When good cholesterol goes bad. Nat Med. 2004; 10: 902–903.[CrossRef][Medline] [Order article via Infotrieve]

46. Marsche G, Levak-Frank S, Quehenberger O, Heller R, Sattler W, Malle E. Identification of the human analog of SR-BI and LOX-1 as receptors for hypochlorite-modified high density lipoprotein on human umbilical venous endothelial cells. FASEB J. 2001; 15: 1095–1097.[Free Full Text]

47. Baldus S, Eiserich JP, Mani A, Castro L, Figueroa M, Chumley P, Ma W, Tousson A, White CR, Bullard DC, Brennan ML, Lusis AJ, Moore KP, Freeman BA. Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration. J Clin Invest. 2001; 108: 1759–1770.[CrossRef][Medline] [Order article via Infotrieve]

48. Eiserich JP, Baldus S, Brennan ML, Ma W, Zhang C, Tousson A, Castro L, Lusis AJ, Nauseef WM, White CR, Freeman BA. Myeloperoxidase, a leukocyte-derived vascular NO oxidase. Science. 2002; 296: 2391–2394.[Abstract/Free Full Text]

This article has been cited by other articles:

				G. Marsche, S. Frank, A. Hrzenjak, M. Holzer, S. Dirnberger, C. Wadsack, H. Scharnagl, T. Stojakovic, A. Heinemann, and K. Oettl Plasma-Advanced Oxidation Protein Products Are Potent High-Density Lipoprotein Receptor Antagonists In Vivo Circ. Res., March 27, 2009; 104(6): 750 - 757. [Abstract] [Full Text] [PDF]

				A. Pirillo, P. Uboldi, C. Bolego, H. Kuhn, and A. L. Catapano The 15-Lipoxygenase-Modified High Density Lipoproteins 3 Fail to Inhibit the TNF-{alpha}-Induced Inflammatory Response in Human Endothelial Cells J. Immunol., August 15, 2008; 181(4): 2821 - 2830. [Abstract] [Full Text] [PDF]

				W.-H. Kan, J.-T. Hsu, Z.-F. Ba, M. G. Schwacha, J. Chen, M. A. Choudhry, K. I. Bland, and I. H. Chaudry p38 MAPK-dependent eNOS upregulation is critical for 17{beta}-estradiol-mediated cardioprotection following trauma-hemorrhage Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2627 - H2636. [Abstract] [Full Text] [PDF]

				J. Lessig, J. Schiller, J. Arnhold, and B. Fuchs Hypochlorous acid-mediated generation of glycerophosphocholine from unsaturated plasmalogen glycerophosphocholine lipids J. Lipid Res., June 1, 2007; 48(6): 1316 - 1324. [Abstract] [Full Text] [PDF]

				G. Marsche, M. Semlitsch, A. Hammer, S. Frank, B. Weigle, N. Demling, K. Schmidt, W. Windischhofer, G. Waeg, W. Sattler, et al. Hypochlorite-modified albumin colocalizes with RAGE in the artery wall and promotes MCP-1 expression via the RAGE-Erk1/2 MAP-kinase pathway FASEB J, April 1, 2007; 21(4): 1145 - 1152. [Abstract] [Full Text] [PDF]

				A. Kontush and M. J. Chapman Functionally Defective High-Density Lipoprotein: A New Therapeutic Target at the Crossroads of Dyslipidemia, Inflammation, and Atherosclerosis Pharmacol. Rev., September 1, 2006; 58(3): 342 - 374. [Abstract] [Full Text] [PDF]

				K. R. Wildsmith, C. J. Albert, D. S. Anbukumar, and D. A. Ford Metabolism of Myeloperoxidase-derived 2-Chlorohexadecanal J. Biol. Chem., June 23, 2006; 281(25): 16849 - 16860. [Abstract] [Full Text] [PDF]

				T. Hasegawa, E. Malle, A. Farhood, and H. Jaeschke Generation of hypochlorite-modified proteins by neutrophils during ischemia-reperfusion injury in rat liver: attenuation by ischemic preconditioning Am J Physiol Gastrointest Liver Physiol, October 1, 2005; 289(4): G760 - G767. [Abstract] [Full Text] [PDF]

				S. J. Nicholls and S. L. Hazen Myeloperoxidase and Cardiovascular Disease Arterioscler. Thromb. Vasc. Biol., June 1, 2005; 25(6): 1102 - 1111. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement Correction (v25,p264) All Versions of this Article: 24/12/2302    most recent 01.ATV.0000148703.43429.25v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marsche, G. Articles by Malle, E. Search for Related Content PubMed PubMed Citation Articles by Marsche, G. Articles by Malle, E. Related Collections Lipids Pathophysiology Other arteriosclerosis Lipid and lipoprotein metabolism Oxidant stress