since these are the leading causes of death in the developed countries , a prevention of atherosclerosis is very crucial . in humans , many defense mechanisms exist against the development of atherosclerosis , and apolipoprotein a - i ( apoa - i ) , the major protein component of high - density lipoprotein ( hdl ) , is associated with some of these mechanisms . antioxidant [ 2 , 3 ] and anti - inflammatory abilities [ 2 , 4 , 5 ] and cholesterol efflux capacity [ 6 , 7 ] are widely recognized as antiatherosclerotic functions of apoa - i . cholesterol efflux plays a particularly important role in the prevention of progression of atherosclerotic lesions since excessive lipids , such as cholesterol , are removed from the atherosclerotic lesions by apoa - i via the atp - binding cassette transporter a1 ( abca1 ) . recently , studies have reported that the cholesterol efflux capacity is inversely associated with the occurrence of cardiovascular events [ 810 ] . it is known that apoa - i , which plays a central role in cholesterol efflux capacity , is modified by various reactions such as oxidation , glycation , and homocysteinylation [ 1115 ] . these modifications of apoa - i , presumed to lead to the development of atherosclerosis , have been the focal point of many studies investigating dysfunctionalities in cholesterol efflux . actually , there are various studies on the relationship between modification of apoa - i and cholesterol efflux capacity that have been reported [ 1618 ] . however , the assay for measurement of cholesterol efflux capacity has not been optimized even by using the same cell line and it is not known whether cellular conditions could lead to a difference in cholesterol efflux capacity . therefore , in this study , we assessed the cholesterol efflux capacity of apoa - i by using various states of cells . moreover , we focused on the role of homocysteine ( hcy ) , one of the risk factors for atherosclerosis , in cholesterol efflux [ 19 , 20 ] . hcy is a metabolite of methionine , an essential amino acid , and is converted to methionine and cysteine . a part of hcy is converted to homocysteine thiolactone ( hcyt ) by methionyl - trna synthetase . hcyt is rich in reactivity and is known to bind to lysine residues ( n - homocysteinylation ) of many proteins in normal human plasma such as albumin , hemoglobin , and fibrinogen . we recently reported that n - homocysteinylated apoa - i ( n - hcy apoa - i ) exists in normal human serum in the ratio of 1.07.4% to total apoa - i . however , the effect of n - homocysteinylation of apoa - i on the cholesterol efflux capacity is not yet confirmed . we also evaluated the cholesterol efflux capacity of n - hcy apoa - i using various states of cells and compared the results to that obtained by using normal apoa - i . blood samples were obtained from healthy volunteers after their informed consent , and the study was approved by the ethics committee of tokyo medical and dental university ( number 1441 ) . thp-1 , a human monocyte cell line , was purchased from atcc and maintained in rpmi-1640 ( sigma - aldrich ) containing 10% fetal bovine serum ( fbs ) , 0.1% penicillin / streptomycin , and 0.1% nonessential amino acids at 37c under 5% co2 . thp-1 cells were incubated with rpmi-1640 medium containing 100  ng / ml of phorbol 12-myristate 13-acetate ( pma ; sigma - aldrich ) supplemented with 0.2% bovine serum albumin ( bsa ) in 24-well cell culture plates at a density of 2.5  10  cells / well . for preparation of various states of differentiated cells , the incubation time was varied from 1 to 5 days as indicated in figure 1(a ) . after removing the supernatant , thp-1 cells were loaded with rpmi-1640 containing acetylated low - density lipoprotein ( acldl ) ( 50  g  protein / ml ) , t0901317 ( 1  mmol / l ; enzo life sciences ) , a liver x receptor ( lxr ) agonist for promoting expression of abca1 , h - cholesterol ( 1  ci / ml ; perkinelmer ) , and bsa ( 0.2% ) . the loading time was also varied over 1 to 5 days to generate different stages of foam cells . thp-1 cells were washed thrice with rpmi-1640 medium containing 0.2% bsa before equilibration with rpmi-1640 supplemented with t0901317 ( 1  mmol / l ) and bsa ( 0.2% ) for 18 hours . after removing the supernatant , cells were incubated with apoa - i ( 10  g / ml ) and t0901317 ( 1  mmol / l ) in rpmi-1640 for additional 4 hours . both the media and cell lysates were counted for radioactivity using the liquid scintillation counter . cholesterol efflux capacity was calculated as a percentage of radioactivity in the medium as per the given formula : { h - cholesterol in medium/(h - cholesterol in medium + h - cholesterol in cells ) }   100  percentage of passive diffusion in the case of no acceptor ( apoa - i ) . for microscopic observation , thp-1 cells were differentiated and loaded at a density of 2.5  10  cells / well for different time periods on cover glass ( thermo fisher scientific ) in 24-well cell culture plates . after incubation , adherent cells were washed with phosphate buffered saline ( pbs ) and then fixed with 3.7% formaldehyde solution . after washing with pbs , differentiated cells were stained with hematoxylin solution , and loaded cells after differentiation were stained with hematoxylin solution followed by working solution of oil red o for assessing the extent of foam cell formation . the plates were washed with distilled water and observed under the microscope ( bx53 - 34-fl , olympus ) . a semiquantification of lipid droplets stained by oil red o was performed with imagej ( national institutes of health ) . thp-1 cells were treated with pma in 6-well culture plates ( 1.0  10  cells / well ) for various time periods as described above , and adherent cells were then washed with pbs . after washing , cells were detached gently with a cell scraper and stained with fluorescein isothiocyanate- ( fitc- ) conjugated anti - cd11b mouse igg ( abcam , ab25727 , monoclonal ) ( dilution 1  :  100 ) for 30  min in a dark place at 4c . after washing , cells were filtered using a nylon filter and the concentration was adjusted to approximately 1.0  10  cells / ml . cd11b expression was analyzed by flow cytometry ( epics xl , beckman coulter ) with excitation at 505  nm and emission at 545  nm . samples were controlled by using fitc - conjugated mouse igg as an isotype control ( abcam , ab91365 , monoclonal ) in each day and the percentage of cells which exceeded the maximum fluorescent intensity of isotype control was defined as certain cd11b positive cells . ldl ( 1.0191.063  g / ml ) and hdl ( 1.0631.21  g / ml ) were isolated from serum obtained from healthy subjects by ultracentrifugation , as described previously . next , ldl and hdl fractions were dialyzed against pbs . to purify apoa - i , hdl fraction was delipidated with ethanol / ether 3  :  2  ( v / v ) and applied to s-200-hr ( sigma - aldrich ) column ( 2.5  90  cm ) , equilibrated with 20  mmol / l tris - hcl buffer ( ph 7.4 ) containing 6.8  mol / l urea . the isolated apoa - i fraction was dialyzed against pbs and stored at 20c until use ( supplemental figure  1 in supplementary material available online at http://dx.doi.org/10.1155/2016/9891316 ) . the expression of cd11b after pma treatment for different time periods was evaluated by western blotting as described previously . thp-1 cells were lysed with radioimmunoprecipitation buffer ( ripa buffer ; 50  mmol / l tris - hcl ph 8.0 including 150  mmol / l sodium chloride , 0.5% sodium deoxycholate , and 1.0% nonidet p-40 ( mp biomedicals ) ) containing protease inhibitor cocktails ( roche ) at 4c . the amount of total protein in cell lysates was measured by folin - lowry method . cell lysates were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and transferred onto pvdf membranes ( millipore ) , which were then incubated with 5% skim milk in tris - buffered saline containing 0.05% tween 20 ( washing buffer ) . pvdf membranes were incubated with primary antibodies , anti - cd11b ( abcam , ab52478 , monoclonal ) and anti--actin ( santa cruz , sc-13 - 656 , polyclonal ) . after three washes with washing buffer , the membranes were incubated with peroxidase - labeled secondary antibody ( beckman coulter , im0831 , polyclonal ) . the bands of cd11b and -actin were visualized by ecl prime western blotting detection reagent ( ge healthcare ) . n - homocysteinylation of apoa - i was performed according to the previously reported method with a slight modification . mol / l ) was incubated with or without 10  mmol / l of hcyt ( mp biomedicals ) at 37c for 6 hours . then , samples were dialyzed against pbs . to confirm the production of n - hcy apoa - i , samples treated with cysteamine were isolated by isoelectric focusing and transferred onto pvdf membranes , which were then incubated with primary antibody , anti - apoa - i ( academy biomedical , 11a - g2b , polyclonal ) . after washing thrice , the membranes were incubated with peroxidase - labeled secondary antibodies ( medical and biological laboratories ) . the bands containing apoa - i were visualized with 3,3-diaminobenzidine-4hcl and h2o2 , and semiquantified by cs analyzer . the differences of cholesterol efflux capacities among states of cells were analyzed using kruskal - wallis test followed by a mann  whitney u test with bonferroni correction . comparison of cholesterol efflux capacity between apoa - i and n - hcy apoa - i was evaluated by a mann  whitney u test . the results are expressed as mean + standard deviation ( sd ) . a p value < 0.05 was considered as statistically significant . thp-1 cells differentiated following treatment with pma for 1 to 5 days . to confirm differentiation of the cells at each stage , the morphology of thp-1 cells was assessed by staining with hematoxylin ( figure 1(a ) ) . mild morphological changes were observed at day 1.5 , and most of these were adendritic cells with approximately 20  m size . from day 1.5 to day 2 , dynamic changes in cells were observed . cells grew larger and showed the appearance of dendrite formation corresponding to the increase in stimulation . next , we evaluated the expression of cd11b , an adhesion molecule , on thp-1 cells by flow cytometric analysis ( figure 1(b ) ) . the expression of cd11b was essentially increased in a stimulation with pma on thp-1 macrophages , while the expression on thp-1 monocytes without pma treatment was not almost detected ( data not shown ) . conclusively , expression of cd11b at day 2 was significantly higher than that at day 1 ( p < 0.01 ) and maintained a plateau state ( figure 1(b ) ) . a similar tendency was also observed in the western blotting analysis ( figures 2(a ) and 2(b ) ) . the effect of differentiation of thp-1 cells on cholesterol efflux capacity of apoa - i was evaluated on the condition that the period of foam cell formation was fixed at 1 day . the cholesterol efflux capacities were the highest and the second highest at day 2 and day 1 of differentiation , respectively , and gradually deceased after day 3 ( figure 3 ) . although the period of foam cell formation was fixed for 1 day , total h - cholesterol taken up by the cells before efflux was largely different according to the differentiation periods , the lowest level at day 1 , a peak at day 2 , and gradual decrease after day 3 ( supplementary figure  2(a ) ) . in addition , h - cholesterol mass in the medium after efflux also showed a similar tendency to total h - cholesterol taken up by the cells ( supplementary figure  2(b ) ) . cholesterol efflux capacities expressed in percentage were largely different from the actual efflux of h - cholesterol mass . in contrast , abca1 expression was the highest at day 1 and gradually reduced in parallel with the duration of pma treatment ( supplementary figure  3 ) . in experiments wherein the stage of differentiation of thp-1 cells with pma treatment was fixed for 2 days , thp-1 macrophages were incubated with acldl for 1 to 5 days . the accumulations of intracellular lipid droplets were evaluated using oil red o staining ( figure 4(a ) ) . lipid droplets stained with oil red o were faintly observed from day 1 and dramatically increased depending on the loading period of acldl ( figure 4(b ) ) . subsequently , the cholesterol efflux capacities of apoa - i in each stage of cells were determined . cholesterol efflux capacity was highest for 1 day after loading with acldl and significantly decreased in a loading time - dependent manner ( figure 5 ) . cellular uptake of total h - cholesterol before efflux showed a gradual increase , whereas medium h - cholesterol after efflux was slightly decreased during acldl loading ( supplementary figures  2(c ) and 2(d ) ) . in addition , no significant change in abca1 expression was observed ( supplementary figure  4 ) . using various stages of cells regulated differentiation and foam cell formation , the effect of n - homocysteinylation on cholesterol efflux capacity of apoa - i was evaluated . to confirm the extent of n - homocysteinylation of apoa - i compared to normal apoa - i , n - hcy apoa - i indicates higher isoelectric point ( pi ) depending on the number of attached homocysteine compounds ( figure 6(a ) ) . the ratio of n - hcy apoa - i to total apoa - i on the isoelectric focusing pattern was 51.9  5.9% ( figure 6(b ) ) . cholesterol efflux capacities of apoa - i and n - hcy apoa - i reached those peaks at day 2 and gradually decreased depending on the periods of the treatment with pma ( figure 7(a ) ) . in case of foam cell formation , a significant decrease was observed in cholesterol efflux capacities of apoa - i and n - hcy apoa - i according to the acldl loading ( figure 7(b ) ) . in addition , the cholesterol efflux capacity of n - hcy apoa - i was significantly lower than that of intact apoa - i by differentiation and foam cell formation at day 1 and day 2 , respectively ; however no significant difference was observed after day 3 ( figure 7 ) . cholesterol efflux is essential for the antiatherosclerotic function of apoa - i , and the evaluation of its capacity would provide important information in terms of prevention and diagnosis of atherosclerosis . however , in previous reports assays for measurement of cholesterol efflux capacity of apoa - i and modified apoa - i have been performed using different methods . therefore , the effect of modification of apoa - i on cholesterol efflux capacity is often controversial [ 1618 ] . our aim was to evaluate the variation in cholesterol efflux capacity of apoa - i under various cellular conditions such as differentiation and foam cell formation and to investigate the effect of n - homocysteinylation of apoa - i on cholesterol efflux capacity in these conditions . first , we confirmed the morphological features and expression of cd11b , which is an established differentiation marker of macrophages of thp-1 cells treated with pma . thp-1 cells treated with pma for 2 days showed a behavior similar to macrophages morphologically , including increase in cell volume . it is known that pma induces cd11b expression via protein kinase c delta ( pkc- ) . an increase in the expression of cd11b on thp-1 cells treated with pma for 2 days was observed by flow cytometric and western blotting analysis while the expression of abca1 decreased from day 1 to day 5 ( supplementary figure  3 ) . pma is presumed to have reduced the abca1 gene expression via pkc activation , since pkc was activated with pma as stated above . taken together , these results suggest that variously differentiated cells were prepared . second , we evaluated the effect of differentiation of thp-1 cells on cholesterol efflux capacity . cholesterol efflux capacity reached a peak at day 2 and decreased after day 3 ( figure 3 ) . at day 1 , the uptake of h - cholesterol into cells and levels detected in the medium were low ( supplementary figures  2(a ) and 2(b ) ) . at day 2 , the uptake of intracellular h - cholesterol was about three times as high as at day 1 . on the contrary , after day 3 , both intracellular and medium h - cholesterol were decreased , and the decreased ratios of medium h - cholesterol were slightly larger than those of intracellular h - cholesterol . these results suggest that the increase of the cholesterol efflux capacity indicated as a percentage at day 2 was caused by the differentiation of thp-1 macrophages , and a decrease after day 3 would be determined by the reduced amount of the expression of abca1 , a pivotal transporter in cholesterol efflux to apoa - i . in many studies , the duration of pma stimulation has been inconsistent for the evaluation of cholesterol efflux capacity [ 1618 ] . our results indicate that the optimum period of pma treatment is 2 days if the cholesterol efflux capacity is required to be assessed at the maximum stage . oil red o positive cells , which indicate the formation of lipid droplets , were increased in proportion to days of loading acldl ( figure 4 ) . meanwhile , cholesterol efflux capacity was decreased in a loading time - dependent manner ( figure 5 ) . although there was an increase in the intracellular h - cholesterol over the passage of days , the levels of secreted h - cholesterol in the medium by apoa - i were decreased gradually ( supplementary figures  2(c ) and 2(d ) ) . moreover , no significant change was observed in abca1 expression ( supplementary figure  4 ) . the excessive accumulation of cholesterol ester in cells is reported to inhibit lysosomal acid lipase ( lal ) activity , an enzyme related to the cholesterol metabolism . free cholesterol taken up into cells is esterified by acyl coenzyme - a cholesterol acyltransferase ( acat ) and stored as lipid droplets . since cholesterol ester is hydrolyzed by lal and removed as free cholesterol through abca1 , the inhibition of lal may lead to a reduction in the cholesterol efflux regardless of the degree of abca1 expression on the cells surface . hence , decrease in cholesterol efflux without reduction of abca1 expression may be related to the loss of lal activity caused by the excessive uptake of acldl , including h - cholesterol , and the effect might be reversed in the presence of an acat inhibitor . these results indicate that the optimum period of loading cholesterol is 1 day if the cholesterol efflux capacity has to be assessed at the maximum stage . longer durations of loading cholesterol decrease cholesterol efflux capacity because of excessive cholesterol accumulation . to the best of our knowledge , this study is the first to indicate the relationship between cholesterol efflux capacity and condition of thp-1 cells such as differentiation and foam cell formation , in depth . using these different states of cells , we examined the effect of n - homocysteinylation on cholesterol efflux capacity of apoa - i . n - hcy apoa - i was detected as a more positively charged band than intact apoa - i ( figure 6 ) and was defined as a percentage to total apoa - i , as described previously . the variation in cholesterol efflux capacities of both apoa - i and n - hcy apoa - i using thp-1 cells treated with pma in different time intervals indicates similar trends , as compared to the values that reached the peaks at day 2 and then gradually decreased ( figure 7(a ) ) . however , significant differences were observed in the cholesterol efflux capacities between apoa - i and n - hcy apoa - i at day 1 and day 2 . on the contrary furthermore , cholesterol efflux capacities of both apoa - i and n - hcy apoa - i were also evaluated using thp-1 cells treated with cholesterol loading at different time intervals . similarly , the cholesterol efflux capacities of these two types of apoa - i showed the same behavior , and n - hcy apoa - i indicated significantly lower capacities than apoa - i at day 1 and day 2 after loading . only a few studies have been reported about the relationship between hcy and cholesterol efflux capacity [ 32 , 33 ] . we previously reported that the cholesterol efflux capacity of apoa - i was not significantly reduced with n - homocysteinylation . in the previous study the period of pma stimulation was 3 days , indicating that the cells might be in the state of excessive differentiation to consider the possible effect of n - homocysteinylation . to confirm the effect of n - homocysteinylation on cholesterol efflux capacity of apoa - i , the influence of modification site of apoa - i and its dose response should be investigated . however , our results in this study reiterate the importance of the cell conditions such as the degree of differentiation and foam cell formation when the effects of any modifications of apoa - i , not limited to n - homocysteinylation , on cholesterol efflux capacity are evaluated . further investigations are needed to assess the cholesterol efflux capacity using other monocytes or monocytic cell lines [ 3440 ] . although this study has only examined apoa - i , studies using hdl could be necessary to confirm a precise mechanism . in conclusion , the cholesterol efflux capacities of apoa - i and n - hcy apoa - i vary depending on the state of the cells ; hence , it is important to evaluate the cholesterol efflux capacity in several states of the cells adapted for a purpose .
apolipoprotein a - i ( apoa - i ) , the main protein component of high - density lipoprotein ( hdl ) , has many protective functions against atherosclerosis , one of them being cholesterol efflux capacity . 
 although cholesterol efflux capacity measurement is suggested to be a key biomarker for evaluating the risk of development of atherosclerosis , the assay has not been optimized till date . 
 this study aims at investigating the effect of different states of cells on the cholesterol efflux capacity . 
 we also studied the effect of apoa - i modification by homocysteine , a risk factor for atherosclerosis , on cholesterol efflux capacity in different states of cells . 
 the cholesterol efflux capacity of apoa - i was greatly influenced by the extent of differentiation of thp-1 cells and attenuated by excessive foam cell formation . 
 n - homocysteinylated apoa - i indicated a lower cholesterol efflux capacity than normal apoa - i in the optimized condition , whereas no significant difference was observed in the cholesterol efflux capacity between apoa - i in the excessive cell differentiation or foam cell formation states . 
 these results suggest that cholesterol efflux capacity of apoa - i varies depending on the state of cells . 
 therefore , the cholesterol efflux assay should be performed using protocols optimized according to the objective of the experiment .