HDL3-Mediated Inactivation of LDL-Associated Phospholipid Hydroperoxides Is Determined by the Redox Status of Apolipoprotein A-I and HDL Particle Surface Lipid Rigidity
Relevance to Inflammation and Atherogenesis
Objectives— Small dense HDL3 particles of defined lipidome and proteome potently protect atherogenic LDL against free radical-induced oxidation; the molecular determinants of such antioxidative activity in these atheroprotective, antiinflammatory particles remain indeterminate.
Methods and Results— Formation of redox-active phosphatidylcholine hydroperoxides (PCOOH) and redox-inactive phosphatidylcholine hydroxides (PCOH) was initiated in LDL by free radical-induced oxidation. Human HDL3 inactivated LDL-derived PCOOH (−62%, P<0.01) and enhanced accumulation of PCOH (2.1-fold, P<0.05); in parallel, HDL3 accumulated minor amounts of PCOOH. Enzyme-deficient reconstituted dense HDL potently inactivated PCOOH (−43%, P<0.01). HDL3-mediated reduction of PCOOH to PCOH occurred concomitantly with oxidation of methionine residues in HDL3-apolipoprotein AI (apoAI). Preoxidation of methionine residues by chloramine T markedly attenuated PCOOH inactivation (−35%); by contrast, inhibition of HDL3-associated enzymes was without effect. PCOOH transfer rates from oxidized LDL to phospholipid liposomes progressively decreased with increment in the rigidity of the phospholipid monolayer.
Conclusions— The redox status of apoAI and surface lipid rigidity represent major determinants of the potent HDL3-mediated protection of LDL against free radical-induced oxidation. Initial transfer of PCOOH to HDL3 is modulated by the surface rigidity of HDL3 particles with subsequent reduction of PCOOH to PCOH by methionine residues of apoAI.
Oxidative stress, an emerging risk factor for premature atherosclerosis and cardiovascular disease, mediates formation of proinflammatory proatherogenic oxidized low-density lipoprotein (oxLDL) in the arterial intima.1 Both 1-electron (lipophilic and hydrophilic free radicals) and 2-electron (hypochlorite, peroxynitrite) oxidants contribute to LDL oxidation in vivo.1 Accordingly, oxLDL contains multiple products of free radical-induced lipid peroxidation, including lipid hydroperoxides (LOOH) as primary products, secondary LOOH-derived short-chain oxidized phospholipids (oxPL), and oxidized sterols.1 Significantly, oxLDL can induce a proinflammatory phenotype in arterial wall cells, which contributes to both endothelial dysfunction and the atherogenic process.2
High-density lipoprotein (HDL) protects LDL from oxidative damage by free radicals and therefore exerts antiinflammatory properties. Indeed, HDL particles inhibit accumulation of primary and secondary peroxidation products in LDL.2 Such antioxidative and antiinflammatory effects of HDL are manifested in animal models as attenuated production of reactive oxygen species and diminished expression of endothelial adhesion proteins, and are associated with attenuated atherogenesis.2,3
HDL particles are, however, highly heterogeneous in their structure, metabolism, and biological functions.4 Among the major HDL subpopulations, small, dense, protein-rich HDL3 are distinguished by their proteome and lipidome.5,6 Furthermore, HDL3 exhibit potent capacity to protect LDL from free radical-induced oxidative damage7 and to inhibit oxLDL-induced apoptosis of endothelial cells.8 Atheroprotective properties of small dense HDL3 are consistent with results of clinical and animal studies.9,10 Such antioxidative activity appears to derive from both nonenzymatic and enzymatic components of HDL37; however, its precise molecular features remain indeterminate.
Apolipoprotein AI (apoAI), the major HDL apolipoprotein, may play a central role in HDL-mediated antioxidation, as Met residues 112 and 148 can reduce LOOH into redox-inactive lipid hydroxides (LOH), thereby terminating chain reactions of lipid peroxidation.11,12 In addition, apoAI attenuates LDL oxidation by removal of seeding LOOH molecules from LDL.13 Enzymatic components potentially contributing to antioxidative activity of HDL include paraoxonase 1 (PON1), platelet-activating factor-acetyl hydrolase (PAF-AH or “lipoprotein-associated phospholipase A2”) and lecithin:cholesterol acyltransferase (LCAT),4 all of which were proposed to hydrolyze proinflammatory short-chain oxPL (see 14 for review).14 However, PON1, PAF-AH, and LCAT are weakly reactive toward LOOH.15–18
Recent proteomic studies have demonstrated that small dense HDL3 particles are enriched in apoAI, apoF, apoJ, apoL-I, PLTP, PON1, PAF-AH, and LCAT.5,6 Furthermore, the HDL3 lipidome is deficient in sphingomyelin,6 a structural lipid with positive impact on surface rigidity and negative impact on LCAT activity, thereby implicating surface phospholipids (PL) in the biological activities of HDL. Indeed, the transfer of LOOH from LDL to HDL can occur, either spontaneously or mediated by lipid transfer proteins, directly between lipoprotein PL monolayers.19,20
Our present goal was to dissect the molecular features of the HDL3-mediated protection of LDL from free radical-induced oxidative damage. We reveal that the potent antioxidative activity of HDL3 critically involves the transfer of phospholipid hydroperoxides (PLOOH) from oxLDL to HDL3 in a process dependent on HDL surface lipid rigidity, with subsequent inactivation of LDL-derived PLOOH by Met residues of apoAI.
Our experimental strategy involved (1) isolation of HDL2 (a mixture of HDL2a and 2b subclasses) and HDL3 (a mixture of HDL3b and 3c subclasses) particles by density gradient ultracentrifugation, (2) cooxidation of HDL and LDL by 2,2′-azobis-(2-amidinopropane) hydrochloride (AAPH), an azo-initiator of lipid peroxidation,21 and (3) coincubation of HDL with LDL preoxidized by AAPH. Details of this strategy, and of: (1) reagents and chemicals, (2) blood samples, (3) fractionation and characterization of plasma lipoproteins, (4) preparation of reconstituted HDL (rHDL) and liposomes, (5) determination of phosphatidylcholine hydroperoxides (PCOOH), phosphatidylcholine hydroxides (PCOH), cholesteryl ester hydroperoxides (CEOOH), PL, cholesteryl esters (CE), lysophosphatidylcholine (lysoPC), free fatty acids, and oxidized Met residues of apoAI by HPLC, (6) the capacity of HDL to protect LDL from oxidative damage, (7) the rigidity of liposomal PL monolayers, and (8) the activities of HDL-associated enzymes, are available in the supplemental materials (available online at http://atvb.ahajournals.org). Data presented are means±SD of at least 3 independent experiments with at least 3 independent HDL samples.
Inactivation of PCOOH and CEOOH During LDL+HDL3 Oxidation
Small dense HDL3 attenuated accumulation of PCOOH (the sum of the 18:2/16:0, 20:4/16:0, and 22:6/16:0 molecular species; supplemental Table I) during LDL oxidation by AAPH. Indeed, levels of PCOOH tended to be lower (−58%) in LDL+HDL3 mixtures as compared to LDL oxidized alone after 2-hour incubation and were significantly decreased at 6 hours (−48%, P<0.05; supplemental Figure IA). Furthermore, PCOOH levels in LDL+HDL3 mixtures were reduced as compared to those in LDL and HDL3 oxidized separately (supplemental Table II). In parallel, reduction in PCOOH levels was observed in LDL reisolated from LDL+HDL3 mixtures after oxidation for 2 hours (−65%) and 6 hours (−44%, P<0.05; supplemental Figure IB). During oxidation, small amounts of PCOOH accumulated in reisolated HDL3 (supplemental Figure IC).
Large light HDL2 tended to attenuate levels of PCOOH in LDL+HDL2 mixtures after 2-hour and 6-hour oxidation (−50% and −44% as compared to LDL oxidized alone respectively; n=4, P=ns). In LDL reisolated from LDL+HDL2 mixtures, PCOOH concentrations tended to fall (−62% and −26% at 2 hours and 6 hours, respectively; n=4, P=ns). Accumulation of PCOOH was significantly more pronounced in reisolated HDL2 as compared to that in reisolated HDL3 (6.6-fold at 2 hours and 2.8-fold, P<0.05, at 6 hours; n=4), consistent with greater rates of oxidation in LDL+HDL2 versus LDL+HDL3 mixtures.
CEOOH levels in LDL (the sum of the 18:2 and 20:4 molecular species; supplemental Table I) were diminished in the presence of HDL3. Indeed, CEOOH levels tended to diminish in LDL+HDL3 mixtures as compared to LDL oxidized alone after both 2-hour and 6-hour oxidation (−38% and −26% respectively; supplemental Figure IIA). A marked reduction in CEOOH content in the presence of HDL3 (−50%) was noted in LDL reisolated from mixtures with HDL3 after 2 hours (supplemental Figure IIB). Overall, the effect of HDL3 on CEOOH accumulation in LDL was less potent than that on PCOOH. In parallel, accumulation of CEOOH was detected in reisolated HDL3 (supplemental Figure IIC).
Similarly, CEOOH concentrations tended to decrease in the presence of HDL2 both in LDL+HDL2 mixtures (−35% and −29% after 2 hours and 6 hours, respectively; n=4, P=ns) and in reisolated LDL (−48% after 2 hours; n=4, P=ns). CEOOH accumulation was markedly higher in reisolated HDL2 as compared to reisolated HDL3 (4.0-fold and 4.7-fold difference; n=4) after oxidation for 2 hours and 6 hours (P<0.05), respectively.
Inactivation of PLOOH and CEOOH in Preoxidized LDL
In separate experiments, LDL were preoxidized and subsequently incubated with HDL in the presence of EDTA. After 2-hour incubation, PCOOH levels in LDL+HDL3 mixtures were significantly decreased (−42%, P<0.01; Figure 1A) as compared to oxLDL alone. Even greater effects on PCOOH levels were noted after prolonged (6-hour) incubation (−62%, P<0.01; Figure 1A). Consistent with these data, HDL3 potently inactivated PCOOH in oxLDL reisolated after 0.5-hour incubation (−64%, P<0.01; Figure 1B), an effect which was even more pronounced at 2 hours (−87%, P<0.001) and at 6 hours (−95%, P<0.001; Figure 1B). In reisolated HDL3, slow accumulation of PCOOH was observed (Figure 1C) consistent with the transfer of PLOOH from oxLDL.
HDL2 equally attenuated PLOOH content in preoxidized LDL, albeit less markedly than HDL3 when compared on a total mass basis. Indeed, PCOOH levels were significantly decreased by HDL2 both in LDL+HDL2 mixtures (−25% and −53%, P<0.05, after 2-hour and 6-hour oxidation, respectively; Figure 1A) and in reisolated oxLDL (−72%, P<0.01, −80%, P<0.001, and −90%, P<0.001, after 0.5-hour, 2-hour, and 6-hour oxidation respectively; Figure 1B). In reisolated HDL, PCOOH levels were preferentially elevated (15-fold) in HDL2 as compared to HDL3 after 0.5-hour incubation, and were 5-fold and 4-fold elevated after 2 hours and 6 hours, respectively (Figure 1C).
In striking contrast, no change in CEOOH concentrations was observed in oxLDL+HDL3 mixtures incubated under the same conditions (supplemental Figure IIIA). Likewise, no effect of HDL3 on CEOOH content in oxLDL reisolated after coincubation was observed (supplemental Figure IIIB). Reisolated HDL3 slowly accumulated low CEOOH levels (supplemental Figure IIIC), thereby resembling the pattern of PCOOH accumulation (Figure 1C). Similarly, no effect of HDL2 on CEOOH content of preoxidized LDL was observed (data not shown). However, CEOOH accumulation was considerably more pronounced in reisolated HDL2 than in reisolated HDL3 (approximately 30-fold after 0.5-hour and 2-hour oxidation and 23-fold after 6 hours; n=4, P<0.05).
HDL-Mediated Inactivation of PCOOH: Role of ApoAI
To define the role of apoAI in the HDL-mediated inactivation of PCOOH, rHDL containing only purified apoAI and palmitoyloleoyl phosphatidylcholine (POPC) at a molar ratio of 1.0/77.1 were prepared. Importantly, the size and density of such rHDL closely resemble those of small dense HDL3.22 When such enzyme-deficient, small, dense rHDL were incubated with oxLDL for 2 hours at 37°C, PCOOH levels were significantly decreased (−43%) as compared to those in oxLDL incubated alone (2.37±0.40 versus 3.96±0.95 μmol/L respectively, n=3; P<0.01). Furthermore, such rHDL displayed potent activity in the LDL oxidation assay; indeed, significant reduction (−64%, P<0.05; Table) in the propagation rate of lipid peroxidation in LDL incubated with apoAI/POPC complexes as compared to LDL oxidized alone was observed. ApoAI/POPC rHDL equally induced a pronounced prolongation of the propagation phase (+104%) and a moderate decrease in the maximal diene concentration (−27%; Table). The capacity of such rHDL to protect LDL from oxidative damage was comparable to that of small dense HDL3b +3c isolated from control human plasma5 (−79% decrement in the propagation rate, +57% increment of the propagation phase duration and −27% decrement in the maximal diene concentration when recalculated to equivalent concentration of protein of 10 mg/dL; n=11).
Implication of Met Residues of ApoAI in HDL-Mediated Inactivation of PCOOH
Met sulfoxide-containing forms of apoAI represented 9.5±2.3% (n=9) of total apoAI in freshly isolated HDL3, suggesting that some Met oxidation occurs in the circulation in vivo or during HDL isolation in vitro.12 After incubation of HDL3 in the presence of oxLDL, both redox-active Met residues of apoAI (Met112 and 148) were significantly transformed (by 58% and 79% after 0.5 hours and 2 hours, respectively) into the corresponding sulfoxides (Figure 2A), thereby demonstrating that inactivation of LDL-derived PCOOH by HDL3 was associated with the conversion of apoAI Met into Met(O). Furthermore, concomitant formation of PCOH occurred, which was accelerated by the presence of HDL3. Indeed, levels of PCOH were elevated 2.1-fold in the presence of HDL3 (P<0.05) and were accompanied by significant reduction in PCOOH levels (−59%; Figure 2B; P<0.05), resulting in the 5.6-fold elevated PCOH/PCOOH ratio (0.67 versus 0.12).
To further evaluate the role of Met residues in the capacity of HDL to inactivate PCOOH, chloramine T was used to selectively oxidize apoAI Met112 and Met148.11 Both apoAI Met residues were fully oxidized by chloramine T (data not shown); such HDL3 revealed diminished capacity to inactivate PCOOH, resulting in increased PCOOH levels (+33%; supplemental Figure IVA). In parallel, a minor increase (+10%) in PCOOH was observed in oxLDL reisolated from the mixture of oxLDL and HDL3 pretreated with chloramine T as compared to oxLDL reisolated from the mixture with native HDL3 (supplemental Figure IVB). Marked increment in PCOOH occurred in reisolated HDL3 pretreated with chloramine T as compared to native HDL3 (+350%, P<0.05; supplemental Figure IVC). As a result, levels of PCOH in oxLDL+HDL3 mixtures were significantly decreased (−70%; supplemental Figure IVD) in the presence of HDL3 pretreated with chloramine T as compared to native HDL3, resulting in a 4.2-fold diminished PCOH/PCOOH ratio.
Finally, apoAI Met residues were selectively oxidized with chloramine T in rHDL. The antioxidative activity of such chloramine T-pretreated rHDL was markedly decreased, both in terms of its capacity to decrease LDL oxidation rate and to prolong LDL oxidation (Table). Furthermore, treatment of apoAI/POPC rHDL with chloramine T diminished (−45%) its capacity to inactivate oxLDL-derived PCOOH as compared to nontreated rHDL (PCOOH levels at the end of the incubation, 3.08±0.70 μmol/L versus 2.37±0.40 respectively; n=3).
Implication of HDL-Associated Enzymes in HDL3-Mediated Inactivation of PCOOH
To evaluate the potential role of HDL-associated enzymes, including LCAT, PAF-AH, and PON1, in the inactivation of LDL-derived LOOH, HDL3 was pretreated with a cocktail of enzymatic inhibitors (DFP which inhibits all 3 enzymes, Pefabloc which selectively inhibits PAF-AH, and EDTA which selectively inhibits PON1; supplemental Table III) and then incubated with oxLDL. Pretreatment significantly decreased the activities of HDL-associated LCAT (−50%), PAF-AH (−90%), and PON1 (−99%; supplemental Figure VA). However, no decrease in the capacity of such HDL3 to inactivate LOOH in oxLDL (supplemental Figure VB) or to delay LDL oxidation (data not shown) was observed. Similarly, no significant loss of the capacity of HDL3 to delay the accumulation of conjugated dienes in LDL was noted as a result of the pretreatment of HDL3 with any given single inhibitor (supplemental Table III).
In a separate experiment, HDL3 was preincubated with DTNB to derivatize free SH-groups involved in LCAT and PON1 activities.11,23 Again, such treatment exerted no influence on HDL3 capacity to inactivate preformed LOOH (data not shown). As expected, treatment of HDL3 with DTNB did not cause oxidation of Met residues of apoAI (data not shown). Finally, 10-minute incubation of HDL3 at 56°C significantly decreased PON1 and LCAT activities but did not influence its capacity to delay LDL oxidation (supplemental Table III).
To evaluate the role of hydrolytic enzymes, products of hydrolytic degradation of PC were equally determined. Levels of lysoPC in reisolated LDL oxidized alone and in oxLDL reisolated from oxLDL+HDL3 mixtures after 6-hour oxidation were similar (2.11±1.02 versus 1.75±1.25 μmol/L, respectively; n=3). Furthermore, no difference was observed between lysoPC levels in HDL3 reisolated from LDL+HDL3 mixtures after 6-hour oxidation and nonoxidized HDL3 (1.00±0.44 versus 0.97±0.31 μmol/L respectively; n=3). Finally, after 6-hour incubation, no accelerated accumulation of free fatty acids was noted in LDL+HDL3 mixtures versus LDL alone (40.0±4.1 versus 43.5±4.9 μmol/L respectively; n=3).
Implication of Surface PL in the HDL-Mediated Inactivation of PCOOH: Role of Acceptor Monolayer Rigidity
To elucidate whether PCOOH could be transferred to acceptor particles from oxLDL, oxLDL was incubated with LOOH-free PC liposomes prepared from nonoxidable POPC. Indeed, PCOOH accumulation in the liposomes was observed with concomitant decrease in PCOOH content of oxLDL (−50%, P<0.001; Figure 3A). To investigate whether such PCOOH transfer depended on physical characteristics of the acceptor PL monolayer, liposomal membrane rigidity was varied by adding sphingomyelin (SM) or free cholesterol (FC). On addition of 30 mol% SM to POPC, transfer of PCOOH to acceptor liposomes was diminished by −10%; addition of 10 and 30 mol% FC decreased PCOOH transfer from oxLDL by −20% and −25%, respectively (Figure 3A). As a result, the rigidity of the liposomal PL monolayer, assessed as generalized polarization (GP) of Laurdan fluorescence, was strongly and negatively correlated with PCOOH transfer (r=−0.98, P=0.02; Figure 3B).
LDL was then oxidized in the presence of small dense rHDL containing either apoAI/POPC, apoAI/POPC/FC, or apoAI/ (POPC+SM). The propagation rate of LDL oxidation was significantly higher in the presence of apoAI/POPC/FC and apoAI/POPC/SM rHDL (+53%, P<0.05, and +46%, P<0.01, respectively; Table) as compared to LDL oxidized in the presence of apoAI/POPC rHDL. In parallel, the propagation phase was markedly shortened in the presence of apoAI/POPC/FC and apoAI/POPC/SM rHDL −113%, P<0.07, and −106%, P<0.06, respectively; Table). By contrast, no difference in the maximal diene concentration was observed between different rHDL preparations, consistent with data for plasma HDL known to influence the rate, rather than the total amount, of LOOH accumulation under the oxidative conditions employed.7
Kinetic Analysis of LDL and HDL Oxidation
Kinetic analysis of LDL and HDL oxidation performed according to Bowry and Stocker21 revealed that the lipid peroxidation rate (Rp) in LDL was decreased by 55% as a result of the addition of HDL3 (supplemental Table IV). The lipid peroxidation rate of HDL3 was markedly (up to 21-fold) lower as compared to those for LDL. Importantly, the sum of Rp values obtained for LDL and HDL3 oxidized the same mixture was lower as compared to the value of Rp obtained for LDL oxidized alone, indicative of the protective effect of HDL toward LDL oxidation.
Our present investigations have identified 2 key determinants of the potent capacity of physicochemically-defined HDL3 particles to protect LDL from oxidative damage by free radicals. Firstly, the HDL PL monolayer, which ensures the transfer of PLOOH from LDL to HDL3 in a process modulated by HDL surface lipid rigidity, and secondly apoAI, which reduces PLOOH to the corresponding redox-inactive PLOH by virtue of Met residues. By contrast, our data does not support the contention that HDL-associated enzymes (ie, PON1, PAF-AH, and LCAT) contribute significantly to the inactivation of LDL-derived LOOH.
We have established that the rigidity of the PL monolayer of HDL particles is a key modulator of the transfer efficiency of PCOOH from LDL to HDL. Indeed, the strong negative correlation of the PLOOH transfer rate to liposomes with membrane rigidity emphasizes the importance of both physical and chemical properties in this process. Furthermore, both the PLOOH transfer rate from oxLDL and the capacity of HDL to delay LDL oxidation decreased in parallel with surface lipid rigidity in rHDL.
The spontaneous translocation of PL-derived LOOH between erythrocytes and LDL is established.24 LOOH transfer equally occurs between plasma lipoproteins in systems consisting of donor/acceptor vesicles and rHDL.19,20 Lipid transfer proteins, including cholesteryl ester transfer protein (CETP), can accelerate the transfer of both PLOOH and CEOOH between plasma lipoproteins.19,20 As HDL and LDL are the major vehicles for transport of PL and CE in plasma, exchange of LOOH with other lipoproteins (eg, VLDL) should be of secondary importance. Clearly then, the transfer of LDL-associated PLOOH to HDL constitutes a key step in the HDL-mediated attenuation of LDL lipid peroxidation.
Consistent with the earlier data of Stocker et al,11,12 HDL3-associated apoAI inactivated LDL-derived PLOOH with reduction to the corresponding PLOH; apoAI methionine residues 112 and 148 presumably constitute the site of such reduction.11,12 Indeed, concentrations of redox-active Met residues in apoAI and of PLOOH in our assay ranged from 1.9 to 4.5 μmol/L and from 1.2 to 4.7 μmol/L, respectively, consistent with a 1:1 reaction stoichiometry. Kinetic analysis revealed that lipid peroxidation rate of HDL3 was markedly lower as compared to that of LDL despite similar molar content of oxidizable fatty acids in HDL3 and LDL.6,25 Furthermore, the sum of lipid peroxidation rates in LDL and in HDL3 oxidized in the mixture was considerably lower than that of LDL oxidized alone under identical conditions. As simple inhibition of oxidation by the addition of less oxidizable substrate can be excluded, these data provide direct evidence for HDL3 as a site of the inactivation of LDL-derived LOOH on exposure to oxidative stress.
Oxidation of specific Met residues in apoAI and apoAII to Met(O) plays a major role in the two-electron reduction of PCOOH and CEOOH in HDL particles.11,12 In addition, lipid-free apoAI can prevent formation of LDL-derived oxidized PL by the removal of LOOH from LDL or arterial wall cells13; LOOH reduction by apoAI Met residues may contribute significantly to such an antioxidative effect. Importantly, rHDL containing only purified apoAI and POPC, but devoid of enzymatic components, and authentic small, dense HDL3b +3c, were comparable in their capacities to delay lipid peroxidation of LDL. ApoAI therefore constitutes the central element in the HDL3-mediated protection of LDL from free radical-induced oxidation. This conclusion is consistent with the potent capacity of HDL3 to inactivate LOOH as HDL3 is enriched in apoAI as compared to HDL2.6
The dissociation of PON1 activity from the capacity of HDL to inactivate LOOH indicates that HDL-associated PON1 does not contribute significantly to the inactivation of LDL-derived LOOH. We and others11,26 demonstrated the existence of PON1-independent antioxidative activity of HDL. The major activity of PON1 has been recently demonstrated as that of a lactonase rather than a peroxidase17; the affinity of PON1 for LOOH is several orders of magnitude lower than its affinity for lactones.17,27 Together with our present data, these findings demonstrate that LOOH do not constitute a significant substrate of PON1 and that the previously proposed mechanism for the PON1-mediated attenuation of LDL lipid peroxidation via hydrolysis of LOOH is not tenable. We hypothesize therefore that the established antiatherosclerotic properties of PON128 are unrelated to its capacity to inactivate LOOH, but rather involve its major activity as a lactonase through a still unknown pathway upstream of the regulation of systemic oxidative stress.
In a similar manner, plasma PAF-AH readily hydrolyzes PAF-like oxPL29; furthermore, PAF-AH can hydrolyze PLOOH,18 leading to the formation of lysoPC and a fatty acid hydroperoxide. In our studies, inhibition of PAF-AH by Pefabloc was without effect on the HDL-mediated inactivation of LDL-derived LOOH, thereby indicating that PAF-AH cannot account for this activity. LCAT may equally hydrolyze short-chain oxidized PC generated during lipoprotein oxidation,15 but is inactive toward oxidized CE, the most abundant oxidized lipid species in LDL.1 Data herein demonstrate that LCAT is at most a minor factor in LOOH inactivation.
We propose a 2-step mechanism for the HDL3-mediated protection of LDL against oxidative damage by 1-electron oxidants (Figure 4). Initially PLOOH is transferred from LDL to HDL3; such transfer is governed by the rigidity of the surface monolayer of HDL, decelerates with increasing rigidity, and can be facilitated by lipid transfer proteins, such as CETP.19 Subsequently, reduction of PLOOH by redox-active Met residues of apoAI results in the formation of PLOH and methionine sulfoxides. Preferential degradation of PCOOH by HDL as compared to CEOOH is consistent with the PCOOH location in the surface monolayer of LDL, resulting in their easier accessibility for transfer to HDL.24 Nonetheless, accumulation of CEOOH in LDL can be inhibited by HDL on LDL+HDL cooxidation as a consequence of decreased accumulation of PLOOH and shorter chain length of lipid peroxidation. The minor role of HDL-associated enzymes in this mechanism is consistent with their inability to supply reductive equivalents for LOOH reduction.
Dense HDL3 was distinct in accumulating lower levels of PCOOH and CEOOH and inactivating lipid hydroperoxides more potently than HDL2. According to our proposed mechanism, the elevated capacity of HDL3 to incorporate or to inactivate (P)LOOH may derive from (1) depletion of sphingomyelin, (2) enrichment in apoAI, or (3) altered conformation of apoAI as compared to HDL2.4,6
The pathophysiological relevance of (P)LOOH inactivation by HDL is highlighted by prooxidative and proinflammatory properties of LOOH.1 In the arterial intima, free radical-induced peroxidation of LDL lipids results in the formation of LOOH as primary products.1 We used AAPH, a well-characterized azo-initiator of oxidation, to model free radical-induced LDL oxidation which involves LOOH formation as a major step.21 Furthermore, the HDL to LDL ratio used in our experiments was close to that in the interstitial fluid.30 Together with glutathione peroxidase (GPx), apoA-I represents a key LOOH-reducing protein in human plasma.31 LOOH reduction mediated by apoAI might therefore be especially relevant in microenvironments depleted of low-molecular-weight compounds, including glutathione and GPx, such as the arterial intima.
The capacity of HDL3 to protect LDL from free radical-induced oxidative damage is deficient in the atherogenic dyslipidemias of type 2 diabetes and metabolic syndrome.14 Elevation in the rigidity of the PL monolayer or deficiency of apoAI may underlie this observation. We therefore propose that induction of selective increase in the concentration of functional HDL3 particles displaying decreased surface rigidity and rich in apoAI may constitute an efficacious therapeutic approach to attenuate oxidative damage, inflammation, and atherosclerosis in dyslipidemic subjects at high cardiovascular risk.
Sources of Funding
These studies were supported by National Institute for Health and Medical Research (INSERM), Agence Nationale de Recherche (France; project COD 2005 Lisa) and the Fondation pour la Recherche Médicale. A.Z. gratefully acknowledges support from Nouvelle Societé Française d'Atherosclérose, MSD and Schering-Plough (France). M.J.C. and A.K. acknowledge the award of a Contrat d'Interface from Assistance Publique - Hôpitaux de Paris/INSERM (France).
Received June 11, 2009; revision accepted September 9, 2009.
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