high - density lipoprotein ( hdl ) is known to have protective effects against the development of atherosclerosis by removing cholesterol from peripheral tissues following delivery to the liver and excretion in bile , a process called reverse cholesterol transport ( rct ) [ 1 , 2 ] . in addition , hdl has antioxidant ability  the ability to protect against the oxidation of low - density lipoprotein ( ldl ) [ 1 , 3 , 4]and can inhibit monocyte migration to subendothelial space [ 1 , 5 , 6 ] . therefore , structural changes in hdl would induce functional changes that could be followed by increased risk of cardiovascular disease . one mechanism that could be connected with the formation of dysfunctional hdl is oxidation induced by myeloperoxidase ( mpo ) released from macrophages in atherosclerotic lesions . mpo catalyzes the chlorination or nitration of tyrosyl residues of apolipoprotein a - i ( apoa - i ) molecules in hdl [ 79 ] . when the tyrosyl residues of apoa - i and apoa - ii are changed to radical form , the initial product of tyrosine oxidation by mpo , some apoa - i and apoa - ii form heterodimers [ 1114 ] . we previously observed that the levels of apoa - i / apoa - ii heterodimers were significantly higher in plasma from patients with acute myocardial infarction compared to control patients . various reports have described the relationship between mpo - oxidized apoa - i and rct . mpo was shown to reduce the rct function of apoa - i [ 1517 ] , and apoa - i chlorination by mpo impaired abca1-dependent cholesterol transport . however , the detailed mechanism by which mpo affects rct by oxidation of apoa - i has not been fully elucidated [ 14 , 19 , 20 ] . in addition , the antioxidative potential of hdl containing apoa - i / apoa - ii heterodimers is unknown . therefore , we further investigated how oxidation of mpo affects the antiatherogenic properties of hdl , such as antioxidant ability , anti - inflammatory ability , and cholesterol efflux capacity . these results could improve our understanding of how quantitative estimates of mpo oxidation ( e.g. , by measurement of apoa - i / apoa - ii heterodimers or by nitrated or chlorinated apoa - i ) can reflect the pathogenesis of lesions . unless otherwise stated , all reagents were purchased from wako pure chemical ( tokyo , japan ) . the study was approved by the ethics committee of tokyo medical and dental university ( number 1441 ) . goat anti - human apoa - i and apoa - ii polyclonal antibodies were purchased from academy biomedical ( houston , tx ) . horseradish peroxidase- ( hrp- ) conjugated rabbit anti - goat igg polyclonal antibody was purchased from medical and biological laboratories ( nagoya , japan ) . biotinylated mouse anti - human apoa - i monoclonal antibody was purchased from mabtech ( cincinnati , oh ) . ldl ( 1.006 < d < 1.063  g / ml ) and hdl ( 1.063 < d < 1.21  g / ml ) were isolated from pooled human serum by ultracentrifugation as described previously . isolated ldl and hdl were dialyzed against phosphate buffered saline ( pbs ) and stored at 80c and 4c , respectively , and hdl was used within 5 days . briefly , proteins separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) , using 1016% polyacrylamide gel under reducing conditions , were transferred to polyvinylidene fluoride ( pvdf ) membranes ( millipore , bedford , ma ) . the membranes were incubated with anti - apoa - i and anti - apoa - ii polyclonal antibodies , respectively , followed by incubation with hrp - conjugated rabbit anti - goat igg . finally , the bands containing apoa - i or apoa - ii were visualized with 3,3-diaminobenzidine tetrahydrochloride and hydrogen peroxide . the relative amounts of apoa - i / apoa - ii heterodimer were analyzed with a cs analyzer ( atto , japan ) . five milliliters of hdl ( 10  mg protein / ml in pbs ) was incubated with 5  ml of 50  mmol / l phosphate buffer ( ph 7.4 ) containing 0.2  mmol / l of hydrogen peroxide and diethylenetriaminepentaacetic acid ( dtpa ) each , 0.4  mmol / l of l - tyrosine , and 20  nmol / l ( unless otherwise stated ) of mpo at 37c for 1  hr and 24  hrs . ldl oxidation induced by copper sulfate ( cuso4 ) was evaluated by the formation of conjugated dienes . ldl ( 50  g protein / ml ) was incubated with cuso4 ( 1  mol / l ) at room temperature in the absence or presence of hdl ( 50  g protein / ml ) , and the absorbance at 234  nm was monitored in 10  min intervals . the lag time was calculated from the kinetics as the intersection of the lag phase and the propagation phase lines . antioxidant ability of hdl was defined as relative prolongation of the lag time in the presence of hdl compared to ldl alone . thp-1 , a human monocyte cell line , was obtained from atcc ( manassas , va ) and propagated in rpmi 1640 at a density of 1  10  cells / ml ; the medium was supplemented with 10% fetal calf serum ( fcs ) containing penicillin ( 100  u / ml ) and streptomycin ( 100  g / ml ) , and propagation took place at 37c in 5% co2 . the cells were then washed extensively with serum - free rpmi 1640 medium , diluted to the appropriate density , and used in the experiments as indicated below . human umbilical vein endothelial cells ( huvecs ) were purchased from lonza japan ( tokyo ) and cultured according to the manufacturer 's instructions . cells were cultured on gelatin - coated culture flasks in egm-2 medium ( lonza ) supplemented with 2% fcs . confluent cultures at passage level 3 were washed with hank 's balanced solution , trypsinized , resuspended in complete media ( as described above ) , and plated onto 24-well gelatin - coated plates at a density of 4  10  cells / ml ( 500  l / well ) . after 48  hrs , cells were treated overnight with media containing 2% lipoprotein - deficient serum , which was separated from fcs as the bottom fraction ( d > 1.210  g / ml ) by ultracentrifugation . a thp-1 cell migration experiment was performed as described previously , using a chemotaxicell containing polycarbonate filters ( 8  m pore size ; kurabo , tokyo , japan ) . the lower chambers were filled with 500  l of the conditioned media obtained from the postculture medium of huvecs for 16  hrs at 37c with or without lipopolysaccharide ( lps ) ( 25  ng / ml ) in the absence or presence of hdl ( 50  g / ml ) and mpo - treated hdl ( 50  g / ml ) , of which monocyte chemotactic protein-1 ( mcp-1 ) concentrations were measured using the elisa kit ( r&d systems , inc . then , the upper chambers were filled with 200  l of culture medium containing 2  10 thp-1 cells . after 28  hrs of incubation at 37c in 5% co2 , thp-1 cells that migrated to the lower chambers were counted on a burker - turk hemocytometer using an inverted microscope . the thp-1 cell migration rate in the absence of lps and hdl was defined as 1 . thp-1 cells were grown in rpmi 1640 , 10% fcs , 0.1% penicillin / streptomycin , and 0.1% nonessential amino acids at 37c under 5% co2 . three days prior to the experiment , cells were differentiated into macrophages by addition of phorbol 12-myristate 13-acetate ( pma : 100  ng / ml ; sigma ) . then , macrophages were loaded with 50  g / ml acetylated ldl and 1  ci / ml [ h]-cholesterol in rpmi 1640 containing 0.2% fcs . after 24  hrs , the labeling medium was removed and cells were washed three times with rpmi 1640 containing 0.2% fcs and then equilibrated in the same media for 18  hrs . cholesterol efflux was assessed for 4  hrs using the media in the presence of 50  g / ml hdl , mpo - treated hdl , or no acceptor . radioactivity in the media and total cell - associated radioactivity were determined by scintillation counting . the percentage of cholesterol efflux was calculated as [ h]-cholesterol in medium/([h]-cholesterol in medium + [ h]-cholesterol in cells )  100 . hdl treated with or without 20  nmol / l mpo for 1 and 24  hrs was analyzed by sds - page under reducing conditions using 10% gel , followed by staining with coomassie brilliant blue r-250 ( cbb ) ( figure 1(a ) ) and by immunoblotting using anti - apoa - i and anti - apoa - ii antibodies ( figure 1(b ) ) . the cbb staining showed almost no differences in any hdl samples , except for 37  kda bands , which tend to increase by mpo treatment . the 37  kda bands were confirmed to react with both anti - apoa - i and anti - apoa - ii antibodies and showed a tendency to be increased by mpo treatment in a time - dependent manner ( figure 1(c ) ) . apoa - i - containing complexes at approximately 50 and 80  kda were also induced by mpo treatment . the bandwidth of apoa - i and apoa - ii monomers ( 28 and 8.5  kda , resp . ) mpo was also confirmed to have no apparent effects on hdl particle size on the native page patterns ( figure 2 ) . the kinetics of ldl oxidation by cuso4 were analyzed in the presence of hdl treated with or without mpo for 1 and 24  hrs ( figure 3(a ) ) . although obvious differences between the presence and the absence of hdl were observed in the monitoring profiles , no significant differences were induced by mpo treatment of hdl . the lag times , which were used to quantitatively estimate antioxidant ability , were significantly prolonged by addition of hdl , at a similar rate compared to that of ldl alone ( figure 3(b ) ) . the culture medium of huvecs stimulated by incubation with lps at 37c for 16  hrs in 5% co2 accelerated the migration of thp-1 cells to a level twofold higher than that of the control ( without lps ) ( figure 4(a ) ) . however , the coexistence of mpo - untreated hdl with lps suppressed the accelerated migration by 90% . in contrast , mpo - treated hdl exhibited a significant ( 70% ) reduction in suppressive capacity compared to the untreated hdl . the culture medium of huvecs containing only untreated hdl showed a tendency to suppress thp-1 cell migration compared to mpo - treated hdl without a statistical significance . it was also confirmed that lps failed to have a direct influence on thp-1 cell migration ( data not shown ) . mcp-1 concentrations in the culture media of huvecs clearly reflected in the levels of migration of thp-1 cells ( figure 4(b ) ) . hdl treated with mpo at 0 , 1 , or 5  nmol / l for 1  hr was analyzed by sds - page followed by immunoblotting using anti - apoa - i ( figure 5(a ) ) and anti - apoa - ii ( figure 5(b ) ) antibodies . a dose - dependent increase in apoa - i / we incubated [ h]-labeled cholesterol - loaded thp-1 macrophages with this mpo - treated hdl to evaluate the capacity for cholesterol efflux ( figure 5(c ) ) . although sds - page profiles of apoa - i and apoa - ii were apparently changed by mpo treatment , there was no effect of mpo treatment at any concentration on the cholesterol efflux capacity of hdl . sds - page followed by cbb staining and immunoblot analysis confirmed that treatment of hdl with mpo stimulated the generation of apoa - i / apoa - ii heterodimers with an apparent molecular mass of 37  kda . bands of approximately 50 and 80  kda , which showed higher intensities than the 37  kda band by immunoblot using anti - apoa - i antibody , could be apoa - i dimers and trimers , respectively . those dimers and trimers might have been formed by tyrosine - tyrosine bonds , since their molecular masses were not reduced by treatment with 2-mercaptoethanol . the intensity and width of apoa - i and apoa - ii monomers in immunoblot patterns tended to increase and to spread , respectively , by mpo treatment . the increasedintensity probably indicates that the monomers were chlorinated by mpo treatment , followed by enhanced reactivity with their respective antibodies , as described previously [ 13 , 27 ] . in addition , the slightly expanded width of the immunoblot bands induced by mpo treatment could indicate the increased molecular mass of the partial apoa - i and apoa - ii by addition of tyrosine and/or chlorine molecules to tyrosine residues . it was also confirmed that mpo did not induce a reconstitution with obvious changes of hdl particle size . no significant difference in antioxidant ability was observed between mpo - treated and mpo - untreated hdl . it means that apoa - i / apoa - ii heterodimers in mature hdl particles did not have an impact on antioxidant ability , which probably indicated that a relatively small number of hdl particles contained apoa - i / apoa - ii heterodimers under both physiological and forced oxidative conditions ( figure 1(b ) ) and that the lipids contained in hdl caused a structural reduction in the oxidative activity of the heterodimers . we previously reported that approximately 2% and 3% of total apoa - i and apoa - ii , respectively , were present as the apoa - i / apoa - ii heterodimer . however , the possibility that nascent hdl , which contains extremely low amounts of lipids , might lose its antioxidant ability when oxidized by mpo is remained . in addition , enzymes attached to hdl , including lcat and paraoxonase-1 , were reported to prevent the oxidation of ldl [ 28 , 29 ] , suggesting that those enzymes  rather than apolipoproteins included in hdl the present study demonstrated that hdl suppressed the stimulation of huvecs induced by lps , followed by the inhibition of monocyte chemotaxis . several studies have reported that hdl neutralizes the effects of lps toxicity [ 3032 ] . these findings indicate that mpo released from macrophages partially impairs the anti - inflammatory ability , the neutralization ability in other words , of hdl . these processes would take place in atherosclerotic plaques and precede atherosclerotic lesions , whereby macrophages are the first inflammatory cells to invade intima and are the main component of plaques . it is known that stimulated endothelial cells release chemokines , such as mcp-1 , fractalkine , and rantes , and induce monocyte migration [ 34 , 35 ] . in a basic study , recombinant mcp-1 directly induced thp-1 migration in a time - dependent manner , but the coexistence of hdl did not inhibit thp-1 cell chemotaxis ( see supplemental figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/592594 ) . our data suggested that hdl could act as a neutralizer against the stimulation of huvecs by lps followed by inhibition of mcp-1 secretion . in conclusion , untreated hdl showed a tendency to suppress thp-1 migration compared to mpo - treated hdl and pbs alone , indicating that mpo treatment reduces an anti - inflammatory potential of hdl through the neutralization of lps . in this cholesterol efflux study , 5  nm mpo - treated hdl showed a modest increase in cholesterol efflux capacity compared with untreated hdl ; however , no significant differences were observed , despite the obvious change in apolipoprotein profiles on sds - page , which suggested that the formation of complexes including apoa - i and/or apoa - ii in hdl did not affect cholesterol efflux capacity . previous studies have suggested that the capacity of commercially available native human hdl to mediate cholesterol efflux was impaired by mpo modulation [ 36 , 37 ] . on the other hand , cholesterol efflux capacity was enhanced by apoa - i / apoa - ii heterodimers induced by oxidation of hdl by tyrosyl radicals . it has also been reported that chlorination of apoa - i decreased cholesterol efflux capacity while nitration of apoa - i did not affect cholesterol efflux , suggesting that the molecular mechanisms responsible for cholesterol efflux capacity might be regulated by delicate structural changes in hdl apolipoproteins , especially apoa - i . it also can not be denied that these differences were induced by the subculture conditions under which thp-1 cells were generated . a previous report indicated that the delicate culture conditions used for thp-1 cells can alter their response to stimulation , suggesting that discrepancies among results arise from differences among experiments . our results suggested that the oxidation of hdl by mpo caused significant reduction of anti - inflammatory potential . therefore , quantitative assays of mpo - oxidized hdl might provide useful information about functional changes in hdl and indicate the levels of locally secreted mpo which reflect the state of atherosclerotic lesions . although many researchers have attempted to use direct measurements of mpo in plasma as a biomarker of atherosclerosis [ 39 , 40 ] , we suggest that a marker such as the apoa - i / apoa - ii heterodimer , which would reflect mpo activity limited to atherosclerotic lesions , is a better indicator of the atherosclerotic state than plasma mpo activity . the plasma mpo activity might not be necessarily proportional to the functional reduction of hdl . this indicates the importance of measuring the materials generated by mpo ( e.g. , apoa - i / apoa - ii heterodimers and nitrated and chlorinated apoa - i ) as markers that reflect the atherosclerotic state , in connection with macrophages , ldl , and hdl in the lesion . further studies are needed to elucidate the actual mechanism behind the contribution of mpo to the development of atherosclerosis and to assess the potential of apoa - i / apoa - ii heterodimers as a biomarker for estimating the anti - inflammatory ability of hdl . the principal effect of mpo oxidation on the antiatherogenic potential of hdl would be the reduction of anti - inflammatory ability . it suggests that measurement of apoa - i / apoa - ii heterodimers , which would reflect mpo activity limited to atherosclerotic lesions , might be useful to estimate anti - inflammatory ability of hdl .
high - density lipoprotein ( hdl ) has protective effects against the development of atherosclerosis ; these effects include reverse cholesterol transport , antioxidant ability , and anti - inflammation . 
 myeloperoxidase ( mpo ) secreted by macrophages in atherosclerotic lesions generates tyrosyl radicals in apolipoprotein a - i ( apoa - i ) molecules , inducing the formation of apoa - i / apoa - ii heterodimers through the tyrosine - tyrosine bond in hdl . 
 functional characterization of hdl oxidized by mpo could provide useful information about the significance of apoa - i / apoa - ii heterodimers measurement . 
 we investigated the effects of mpo - induced oxidation on the antiatherogenic functions of hdl as described above . 
 the antioxidant ability of hdl , estimated as the effect on ldl oxidation induced by copper sulfate , was not significantly affected after mpo oxidation . 
 hdl reduced thp-1 monocyte migration by suppressing the stimulation of human umbilical vein endothelial cells induced by lipopolysaccharide ( lps ) . 
 mpo - oxidized hdl also showed inhibition of thp-1 chemotaxis , but the extent of inhibition was significantly attenuated compared to intact hdl . 
 mpo treatment did not affect the cholesterol efflux capacity of hdl from [ 3h]-cholesterol - laden macrophages derived from thp-1 cells . 
 the principal effect of mpo oxidation on the antiatherogenic potential of hdl would be the reduction of anti - inflammatory ability , suggesting that measurement of apoa - i / apoa - ii heterodimers might be useful to estimate anti - inflammatory ability of hdl .