atherosclerosis , one of the major causes of morbidity and mortality in the western world , involves complex interactions among endothelial cells of the arterial wall , blood cells , and circulating lipoproteins . oxidative stress , which is mainly derived from mitochondrial dysfunction , decreases nitrous oxide ( no ) synthesis , upregulates the secretion of adhesion molecules and inflammatory cytokines , and is responsible for the oxidation of low - density lipoproteins ( ldls ) [ 2 , 3 ] . these events play a key role in the pathogenesis of atherosclerosis [ 4 , 5 ] . paraoxonase-1 ( pon1 ) is an enzyme found in the circulation associated with high - density lipoproteins ( hdls ) [ 6 , 7 ] . the original function attributed to pon1 was that of a lactonase , and lipophylic lactones constitute its primary substrates . pon1 also degrades oxidized phospholipids and , as such , plays a role in an organism 's antioxidant system . in the atherosclerosis process , pon1 accumulates in the artery wall , and pon1 mice have been shown to have greater levels of oxidized ldl and larger atheromatous plaques when fed a proatherogenic diet . pon1 also inhibits the production of the proinflammatory chemokine monocyte chemoattractant protein-1 ( mcp-1 ) , induced by oxidized ldl in endothelial cells .    despite its potential clinical and biochemical relevance , there is a paucity of studies investigating the influence of pon1 on metabolic alterations when oxidized ldl is incubated with endothelial cells . we reasoned that metabolomics might be a useful tool to evaluate the effects of this enzyme . the study was complemented with an evaluation of oxidative stress and apoptosis in this cell line . we employed primary cultures of human umbilical vein endothelial cells ( huvecs ) , cultured according to the manufacturer 's instructions ( invitrogen , carlsbad , ca , usa ) . huvecs were grown in medium 200 supplemented with low serum growth , 10  mg / l gentamicin and 0.25  mg / l amphotericin ( all these reagents were from invitrogen ) , and maintained in a humidified incubator at 37c , with 5% co2 . , cells were plated in 10  cm petri dishes at a density of 2.5  10 cells per dish , and at passage 3 . petri dishes at 70% confluence were incubated over 24  h with isolated human ldl ( 50  mg / l ) , oxidized ldl ( 50  mg / l ) , oxidized ldl ( 50  mg / l ) + hdl ( 40  mg / l ) from wild type mice , oxidized ldl ( 50  mg / l ) + hdl ( 40  mg / l ) from pon1 mice , or with serum - free media as controls . normal human sera were obtained from healthy individuals participating in a population - based study being conducted in our institution . the study was approved by the ethics committee ( institutional review board ) of the hospital universitari sant joan de reus . sera were pooled and used for lipoprotein fractionation and ldl isolation by sequential preparative ultracentrifugation [ 12 , 13 ] . human oxidized ldl was prepared by incubation of native ldl with 5  m cuso4 , as described previously . increased thiobarbituric acid - reactive substances levels were detected in ldl after oxidation ( 45 versus < 0.5  mmol / g protein ) . wilmington , ma , usa ) , and pon1 mice were the progeny of those provided by the division of cardiology of the university of california in los angeles and were of a c57bl/6j genetic background . animals were housed under standard conditions and given a commercial mouse diet ( 14% protein rodent maintenance diet , harlan , barcelona , spain ) in accordance with our institutional guidelines . at 16 weeks of age , they were sacrificed and approximately 30  ml of sera were pooled for hdl isolation [ 12 , 13 ] .   the metabolomics platform employed in the present study has been previously described in detail . briefly , small molecule metabolites from an equivalent amount of cell cytoplasm homogenates were extracted with methanol , and the resulting extract divided into equal fractions for analysis by ultrahigh performance liquid chromatography - tandem mass spectrometry ( uplc - ms / ms ; separately under positive mode and negative mode ) and gas chromatography - mass spectrometry ( gc - ms ) . metabolites were identified by comparing the ion data obtained to a reference library of ~2,800 chemical standard entries . comparisons included retention times , mass ( m / z ) , and ms or ms / ms spectra . results of metabolomics measurements are expressed as the mean quotients between the areas under the peak of the different experimental conditions . http://cran.r - project.org/.   we analyzed caspase 9 expression in endothelial cell homogenates as a marker of apoptosis pathways . the cytoplasmic homogenates were prepared with a precellys 24 homogenizer ( bertin technologies , montigny - le - bretonneux , france ) . denaturing electrophoresis was performed in polyacrylamide gels ( 412% ) from invitrogen ( carlsbad , ca , usa ) . blotting was performed with the ecl advanced western blotting detection kit ( ge healthcare , fairfield , ct , usa ) using a rabbit anticaspase 9 antibody at 1  :  2000 dilution ( abcam , cambridge , uk ) . cells ( 300  l of cell suspension at approximately 10 cells / l ) were stained with annexin v conjugated with fluorescein isothiocyanate in the presence of propidium iodide . we used the annexin - fitc kit ( beckman - coulter , fullerton , ca , usa ) according to the manufacturer 's instructions , in a coulter epics xl - mlc flow cytometer ( beckman - coulter ) . pon1 lactonase activity in the culture 's supernatant was measured as the hydrolysis of 5-thiobutyl butyrolactone ( tbbl ) , as described . the assay reagent contained 1  mmol / l cacl2 , 0.25  mmol / l tbbl , and 0.5  mmol / l 5,5-dithio - bis-2-nitrobenzoic acid ( dtnb ) in 0.05  mmol / l tris - hcl buffer ( ph = 8.0 ) . activities were expressed as u / l ( 1  u = 1  mmol of tbbl hydrolyzed per minute ) . the concentration of total peroxides in the supernatant was determined by a colorimetric enzymatic assay ( immundiagnostik , ag , bensheim , germany ) . pon1 lactonase activity remained relatively low in supernatants of those cultures not containing added hdl . pon1 lactonase activity significantly increased in those cultures with normal hdl and returned to low levels in those cultures with hdl from pon1 mice . these results were as expected and provide a quality control of the hdl preparations obtained ( figure 1(a ) ) . total peroxide concentrations in the supernatants were maximal in the cultures with added oxidized ldl and showed a significant decrease following the addition of normal hdl . this decrease was not as marked following the addition of hdl from pon1 mice ( figure 1(b ) ) . we analyzed 124 biochemical compounds by nondirected metabolomics , corresponding to carbohydrate , lipid , amino acid , and nucleotide metabolism , as well as vitamins and xenobiotics . the main findings corresponded to carbohydrate and phospholipid metabolism and are summarized in the following sections . the addition of ldl to cultured endothelial cells decreased the levels of gluconate , galactose , and phosphorylated hexose intermediates . these molecules are important entrance intermediates in energy- and biomass - generating pathways such as glycolysis , pentose phosphate , and protein glycosylation . their decreases suggest that these pathways were activated to a greater extent in endothelial cells treated with ldl , compared to control - treated cells . in contrast , increased levels of gluconate , galactose , and phosphorylated hexose intermediates were seen in all cells that were treated with oxidized ldl , relative to ldl alone , and regardless of whether hdl was also added to the cultures ( figure 2 ) . relative to control cultures , the addition of ldl resulted in increased levels of 3-phosphoglycerate and 2-phosphoglycerate ( which are 3-carbon glycolytic intermediates ) . this same treatment also increased the levels of the tca cycle intermediates ( fumarate and malate ) relative to control cultures . an interpretation of these data is that uptake of ldl by endothelial cells results in the generation of acetyl - coa , which drives flux through the tca cycle . increased levels of ldl - generated acetyl - coa may have relieved the need for carbohydrate - derived precursors , thereby inhibiting glycolytic flux into the tca cycle and elevating the 3-carbon intermediates .   by comparison , treatment of endothelial cells with oxidized ldl may have induced levels of oxidative stress that were sufficient to impair normal energy pathways . for example , in cells treated with oxidized ldl , 6-carbon glycolytic intermediates accumulated , whereas the 3-carbon intermediates were reduced . this may be due to changes in glyceraldehyde-3-phosphate dehydrogenase ( gadph ; levels or activity ) in response to oxidized ldl , since superoxide overproduction inhibits gadph through a mechanism that involves poly ( adp - ribose ) polymerase ( parp ) activation . likewise , tca cycle intermediates were lower in oxidized ldl - treated cells due , most likely , to the attenuated conversion of 6-carbon glycolytic intermediates to 3-carbon compounds that feed into this cycle through pyruvate and acetyl - coa . these changes suggest that energy production through glycolysis is impaired , since atp generation occurs downstream of gadph activity . the addition of normal hdl to oxidized ldl - treated cells partially reverses its impact on energy metabolism pathways , since levels of the 3-carbon glycolytic intermediates as well as tca cycle intermediates are more similar to levels observed after ldl treatment alone . it is of note that the impact of addition of hdl from pon1 mice on these molecules was intermediate between the effects produced by treatment with pon1-containing hdl and of no hdl ( figure 3 ) . this observation is of considerable importance , because parp activation and its consequent metabolic changes have been associated with endothelial dysfunction in diseases such as atherosclerosis and diabetes [ 17 , 18 ] . indeed , the levels of circulating endothelial cells are increased in patients with diabetes mellitus , and pon1 has been shown to attenuate diabetes development in mice [ 20 , 21 ] . our results suggest that the beneficial role of pon1 may involve , at least in part , a protection against the biochemical changes leading to endothelial dysfunction . levels of choline , ethanolamine , and glycerol-3-phosphate  key building blocks for phospholipids  were similar in endothelial cells following treatment with ldl , when compared to levels in control cells . by comparison , oxidized ldl reduced levels of phospholipid precursors and increased the levels of at least one phospholipid breakdown product . this could indicate that oxidized ldl induces membrane damage , breakdown , or remodeling . as was observed for the energy metabolism pathways , coadministration of normal hdl to oxidized ldl - treated cells reversed , or partially reversed , these deleterious effects . however , the addition of hdl from pon1 mice only generated subtle changes in phospholipid - related compounds , when compared to treatment with normal hdl ( figure 4 ) . the observation of alterationsin phospholipids levels and the suggested membrane damage channeled us towards investigating the possibility of an increased apoptosis in endothelial cells incubated with oxidized ldl , and a possible protection by introducing hdl as coincubation . the activation of this enzyme is a good indicator of apoptosis induction , since caspase 9 plays a determinant role in apoptosome formation . we observed that oxidized ldl addition increased caspase 9 expression and the percentage of apoptotic endothelial cells , when compared to control cells and cells treated with normal ldl . however , the influence of hdl from pon1 animals was much lower ( figures 5(a ) and 5(b ) ) . we observed a strong direct correlation ( r = 0.91 ; p < 0.001 ) between total peroxides concentrations and the percentage of apoptotic cells ( figure 5(c ) ) . previous studies had shown that increased lipid peroxidation in hdl particles from coronary artery disease patients was associated with an impaired capacity of this particle to stimulate endothelial no production . notably , pon1 has been reported to prevent lipid peroxidation in hdl particles and to promote hdl - mediated inactivation of oxidized lipids in ldl . further , hdl and pon1 decreased the formation of malondialdehyde - like epitopes and the formation of apoptotic particles in monocytes . a very recent study showed that hdl from healthy people induced the expression of endothelial antiapoptotic protein bcl - xl and reduced endothelial cell apoptosis in vitro as well as in vivo in apoe - deficient mice . in contrast , hdl from coronary artery disease patients did not inhibit endothelial apoptosis , failed to activate endothelial bcl - xl , and stimulated endothelial proapoptotic pathways . our findings of a decreased oxidized ldl - induced apoptosis by normal hdl , but not by hdl from pon1 mice , together with a significant association between lipid peroxidation ( as measured by total peroxides concentrations ) and the percentage of the apoptotic cells would tend to confirm this very recent information . our results suggest that pon1 may play a significant role in cell survival by improving mitochondrial function . indeed , mitochondria regulate apoptosis in response to cellular stress signals and , hence , determine whether cells live or die . as such , it is probable that peroxides constitute important candidates in the regulation of cell death , and that mitochondria act as both sensor and effector sites . this could explain the influence of apoptosis - related proteins on mitochondrial respiration . whether or not this finding has any impact on the atherosclerosis process warrants further exploration . epidemiological studies have shown that the risk of atherosclerosis is inversely associated with hdl concentrations . the protective effect of this lipoprotein has been attributed , in part , to the antioxidant and anti - inflammatory action of pon1 . we have showed , previously , that pon1 inhibits mcp-1 induction in endothelial cells , which suggested a protective role against liver inflammation mediated by mcp-1 . more recent studies indicated that the anti - inflammatory effect of pon1 depends on its association with hdl , and that pon1 stimulates hdl antiatherogenicity , and macrophage response , and increases the duration over which hdl is able to prevent ldl oxidation . the present study is novel in using a metabolomic approach to investigate the protective effect of pon1 on endothelial cells incubated with oxidized ldl . these include an impaired glycolysis , tca cycle , phospholipids , and activation of apoptotic pathways . these changes were ameliorated by incubation with normal hdl , while hdl isolated from pon1 mice showed an impaired efficiency to protect against the oxldl - induced changes . these results extend the current knowledge on the protective role of hdl and pon1 against oxidation in endothelial cells .
we studied the influence of pon1 on metabolic alterations induced by oxidized ldl when incubated with endothelial cells . 
 huvec cells were incubated with native ldl , oxidized ldl , oxidized ldl plus hdl from wild type mice , and oxidized ldl plus hdl from pon1-deficient mice . 
 results showed alterations in carbohydrate and phospholipid metabolism and increased apoptosis in cells incubated with oxidized ldl . 
 these changes were partially prevented by wild type mouse hdl , but the effects were less effective with hdl from pon1-deficient mice . 
 our results suggest that pon1 may play a significant role in endothelial cell survival by protecting cells from alterations in the respiratory chain induced by oxidized ldl . 
 these results extend current knowledge on the protective role of hdl and pon1 against oxidation and apoptosis in endothelial cells .