Oleic acid inhibits stearic acid-induced inhibition of cell growth and pro-inflammatory responses in human aortic endothelial cells.

Saturated fatty acids (SFAs), significant components of both enteral/parenteral nutritional formulations (including diet), are linked to cardiovascular disease complications, such as atherosclerosis. We investigated whether oleic acid (C18:1n-9) reduces the growth inhibitory and pro-inflammatory effects of the stearic acid (C18:0) in human aortic endothelial cells (HAEC). Stearic acid induced growth inhibition at concentrations less than 50 μM, whereas higher concentrations invoked cytotoxicity. Stearic acid-induced growth inhibition and cytotoxic effects were eradicated upon cosupplementation with oleic acid (25 μM). Oleic acid (as low as 5 μM) also inhibited the stearic acid-induced increase in intercellular adhesion molecule-1 (ICAM-1) expression. Stearic acid-induced phosphorylation of nuclear factor-kappa B (NF-κB), a transcriptional regulator of ICAM-1, was also reduced by oleic acid. HAECs supplemented with either stearic or oleic acid resulted in cellular incorporation of C18:0 and C18:1n-9, respectively. Stearic acid primarily incorporated into phospholipids without increasing the total fatty acid content in HAECs. In contrast, oleic acid, with or without stearic acid, incorporated into both phospholipids and triglycerides, with a significant increase in total fatty acid amounts in triglycerides. Our data suggest that oleic acid has the ability to reduce the inflammatory effects of long-chain SFAs in HAECs through reducing cellular stearic acid incorporation and NF-κB activation.


HAEC culture
A primary cell line derived from HAEC was maintained in endothelial basal medium-2 (EBM-2) containing 5% FBS and the bullet kit materials as specifi ed by the manufacturer. Cells were maintained at 37°C in a humidifi ed atmosphere in the presence of 5% CO 2 . Only endothelial cell cultures of less than 10 passages and 80-90% confl uence were used in this study.

Growth inhibition and cytotoxicity assay
Endothelial cells (5000 cells/well) were cultured for 6 h in EBM-2 complete medium in 96-well, fl at bottom tissue culture plates to establish a linear growth rate. At this stage, a subset of established wells were used to determine cellular growth (X) (baseline at day 0) by administering WST-1 reagent (10 l/well), whereas medium in other wells was replaced with EBM-2 complete medium supplemented with treatment conditions (100 l total volume/well) for growth inhibition assessment. Cell cultures were maintained for 48 h at 37°C in a humidifi ed atmosphere containing 5% CO 2 . WST-1 reagent was added, and readings were used to calculate growth inhibition or cell loss based on baseline readings. Vehicle controls (EBM-2 supplemented with 2.2% fatty acid BSA) were used to determine total cell growth (Y). The cell growth (Z) was normalized to 100% using the formula [(Y Ϫ X / X) × 100]. Effects of fatty acid supplementation were determined compared to Z values. Values from 100 to 0 indicate cell growth inhibition, whereas values from 0 to Ϫ 100 indicate cell death. Results are expressed as mean ± SD for at least four determinations.

Apoptosis detection
Cells (1.0 × 10 5 ) were plated in EBM-2 complete medium in 6-well tissue culture-treated plates overnight to initiate a linear growth rate. Spent media was replaced with 2 ml of complete EBM-2 supplemented with varying combinations of albuminbound stearic and oleic fatty acids. Stock solutions (1 mM) of stearic and oleic fatty acids were prepared by complexing with BSA as previously described ( 41 ). At the conclusion of the treatments, the cells were rinsed in PBS and trypsinized from the wells. All spent medium, PBS washes, and trypsinized cells were collected and combined to harvest both adherent and nonadherent cells for analysis. Cell pellets were resuspended in Annexin V FLUOS/propidium iodide labeling solution, which was prepared as described by the manufacturer. Cell suspensions were labeled in the dark at room temperature for 20 min. Analysis was performed on a FACSCalibur fl ow cytometer (Becton Dickinson, San Jose, CA) equipped with a 15 mW air-cooled argon-ion laser emitting at a 488 nm wavelength. Annexin V FLUOS was detected through a 530 nm band pass fi lter, while propidium iodide was detected through a 650 nm long pass fi lter. Data were quantifi ed using CellQuest software (Becton Dickinson).

Flow cytometric analysis of adhesion molecule expression
Trypsinized endothelial cells (1 × 10 5 /sample) were washed in PBS containing 0.5% BSA and resuspended into a volume of 100 l of this labeling buffer. Cells were labeled with 0.25 g of phycoerythrin (PE)-conjugated intercellular adhesion molecule-1 (ICAM-1), otherwise known as CD54, antibody for 20 min. Subsequently, the cells were washed twice in PBS containing 0.5% BSA and resuspended into 250 l of the wash buffer. An isotype control was established for each sample set to ensure specifi city of nutritional formulations/diets, the potential for adverse vascular effects initiated by SFAs are cause for clinical concern. These fatty acids may contribute to infl ammation and vascular dysfunction in patients with both acute and chronic diseases.
The Mediterranean diet and olive oil have been associated with a reduced risk of cardiovascular disease ( 31,32 ). The reduction in cardiovascular risk with olive oil occurs despite maintenance of SFA intake ( 33 ). One of the primary fatty acid components of the Mediterranean diet and olive oil is oleic acid, a monounsaturated fatty acid. Carluccio et al. hypothesized that the cardioprotective effects of the Mediterranean diet were a result of oleic acid's ability to inhibit endothelial activation ( 34 ). Their fi ndings provide evidence for oleic acid's ability to diminish endothelial cell activation, irrespective of the stimulus utilized, by suppressing the upregulation of adhesion molecules. In a randomized trial, an oleic acid-rich diet was found to improve endothelial-dependent vasodilation ( 35 ). It has also been reported that oleic acid can protect Chinese hamster ovary cells from palmitate-induced lipotoxicity by promoting triglyceride synthesis within the cells ( 36 ). Gao et al. investigated the protective effect of oleate against palmitate-induced insulin resistance in L6 myotubes and reported that the protective effect was dependent upon a phosphoinositide 3-kinase mechanism ( 37 ). In MDA-MB-231 breast cancer cells, oleate counteracted the ability of SFAs to induce apoptosis ( 38 ). Other investigators have reported the benefi cial effects of cosupplementing SFAs with oleic acid in other cell types ( 36,37,39,40 ).
Because nutritional formulations (both parenteral and enteral) and diets contain signifi cant amounts of longchain SFAs that are potentially harmful to the vasculature of patients with both acute and chronic infl ammatory diseases, the present investigation was undertaken to determine if oleic acid cosupplementation could reduce SFA-induced pro-infl ammatory responses and cytotoxicity. This study characterizes oleic acid's ability to prevent SFAinduced endothelial cell growth inhibition and cytotoxicity, as well as its ability to diminish SFA-induced endothelial cell pro-infl ammatory phenotypes. Furthermore, this study evaluates fatty acid incorporation and compartmentalized within the cell. Our results suggest that oleic acid neutralizes the negative impact of SFAs on endothelial cell functions by displacing stearic acid from the phospholipid bilayer, thereby preventing stearic acid-induced signaling cascades that promote infl ammation and/or induce apoptosis.

Materials
HAEC and the EGM-2MV Bullet kits (endothelial growth medium-2 microvascular) were purchased from Lonza Incorporation (Walkersville, MD). Chemicals and reagents were acquired from Sigma Chemical Co. (St. Louis, MO), unless otherwise noted. Consumable tissue culture materials were obtained from Fisher Scientifi c (Pittsburgh, PA). The cell proliferation reagent WST-1 and the Annexin V FLUOS staining kits were purchased from Roche Applied Science (Indianapolis, IN). All fatty acids follows. Internal standards for phospholipids (1,2 ditricosanoylglycero-3-phosphocholine; C23:0-PL) and triglycerides (tritricosanoin; C23:0-TG) were added to a known volume of cell lysates. Lipids were extracted using the Folch method. The lipids were resolved on silica gel G-coated plates via thin layer chromatography using a benzene/diethyl ether/ethyl-acetate/acetic acid (80:10:10:0.2 v/v) solvent system. Using standards, the phospholipid and triglyceride fractions were identifi ed by spraying the plates with a primulin (0.01% w/v) solution in acetone-water (40:40 v/v) and exposing to an UV light source. The identifi ed bands were scraped from the silica gels, and 2 ml of methanol: benzene (4:1 v/v) was added to each sample tube. The tubes were gently vortexed and sonicated for 5 min in a water bath at room temperature. Tubes were then placed on a dry ice bath for 10 min before the addition of 200 l of acetyl chloride. Samples were purged with nitrogen gas and permitted to acclimate to room temperature. Triglyceride samples were placed in a heating block for 10 min. at 70°C to facilitate methanolysis of the internal standard. Transesterifi cation of all samples occurred at room temperature over a 24-h incubation period. This reaction was neutralized and recovered as previously described. The GC column and conditions are described above.

Oil Red O staining of lipid droplets
Triglycerides and cholesterol esters can be packaged intracellularly as lipid droplets. HAECs were plated on 4-well Permanox chamber slides and supplemented with albumin-bound fatty acids for 24 h. The neutral lipids packaged within the HAECs were stained using an Oil Red O staining kit procedure described by the manufacturer. Cell nuclei were counterstained with modifi ed Mayer's hematoxylin.

Statistical analysis
Data represent the mean ± SD of at least three determinations. Statistical signifi cance between groups of data was determined using Student's t -test. When a calculated P value of less than 0.05 is reported, statistical signifi cance is indicated with an asterisk.

Neutralizing effect of oleic acid on stearic acid-induced HAEC growth inhibition and cytotoxicity
Endothelial cells were treated with varying concentrations of stearic and oleic acid for 48 h under standard culture conditions. Fig. 1 depicts the attenuating effect of oleic acid on stearic acid-induced growth inhibition and cytotoxicity. Stearic acid inhibited growth of HAECs at concentrations up to 50 M. At higher concentrations, stearic acid induced cytotoxicity (i.e., cell death). Cosupplementation of stearic acid with oleic acid, at concentrations as low as 5 M, signifi cantly improved the negative impact of stearic acid. Oleic acid (25 M) cosupplementation negated the growth inhibitory effects of up to 50 M stearic acid. Furthermore, oleic cosupplementation prevented stearic-induced cytotoxicity at higher concentrations. Although oleic cosupplementation prevented the stearic acid-induced cytotoxicity at the 100 M dose, the HAECs were still signifi cantly growth inhibited.

Effect of oleic acid supplementation on stearic acid-induced apoptosis/necrosis
HAECs were treated with stearic and oleic acid for 24 h prior to labeling with Annexin V FLUOS and propidium the antibody binding. Analysis was performed on a FACSCalibur fl ow cytometer (Becton Dickinson) equipped with a 15 mW aircooled argon-ion laser emitting at a 488 nm wavelength. Fluorescence was detected through a 585 nm band pass fi lter and quantifi ed using CellQuest Software (Becton Dickinson). Results indicate the mean fl uorescent intensity of gated endothelial cells, which excluded cellular debris and particles.

Western blot analysis
HAEC cultures were supplemented with albumin-bound fatty acids for 24 h in 2 ml of EBM-2 complete medium in 6-well tissue culture-treated plates. Treated cells were solubilized with 250 l of lysis buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 100 mM NaF, 2 mM Na 3 VO 4 , 10% glycerol, 1% Nonidet P-40, 2 mM PMSF, 1 g/ml leupeptin, 0.15 units/ml aprotinin, and 2.5 mM diisopropyl fl uorophosphate) for 10 min on ice. Detergent-solubilized extracts were centrifuged to remove insoluble matter, and the protein content of each sample was determined using a BCA (bicinchoninic acid) Protein Assay Kit (Pierce, Rockford, IL). Subsequently, 10 g of protein in Laemmli sample loading buffer was loaded onto each lane and separated on a 4-12% polyacrylamide gradient gel. Resolved proteins were electrophoretically transferred onto nitrocellulose membranes (Millipore Corporation, Bedford, MA) for immuno-Western blot analysis. Blots were then incubated in specifi c primary antibodies according to the manufacturer's specifi cations, and proteins were detected using a peroxidase-conjugated secondary antibody (1:2500 dilution in TBS containing 1% Tween 20 and 1% BSA) with an ECL system (Amersham Pharmacia Biotechnology, Piscataway, NJ) as specifi ed by the manufacturer. For reprobing, membranes were stripped in buffer consisting of 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM ␤ -mercaptoethanol for 30 min at 50°C, followed by six washes in TBS containing 1% Tween 20. Stripped blots were blocked and reprobed with an alternative antibody.

Fatty acid incorporation into the endothelial cells
Subconfl uent endothelial cells were cultured for 24 h in EBM-2 complete media either in the presence or absence of fatty acids. After incubation, the cells were trypsinized and washed three times in PBS (calcium-and magnesium-free) containing 0.5% fatty acid-free BSA to ensure removal of free fatty acids. Cell pellets were resuspended in PBS and sonicated to lyse the cells. An internal standard (C23:0) was added at a known volume to the cell lysates. A portion of the remaining lysates was used to determine the protein concentration to quantify fatty acid content in relationship to the protein content.
Lipids were extracted with chloroform-methanol (2:1) using the Folch method ( 42 ). Using a modifi ed procedure from Lepage and Roy, the fatty acids were transesterifi ed in 2 ml of methanol-benzene (4:1, v/v) after cooling the samples in a dry ice bath for 10 min and adding acetyl chloride to each sample ( 43 ). Transesterifi cation occurred at room temperature over a 24-h incubation period. The reaction was neutralized with the addition of a 6% K 2 CO 3 solution. The samples were vortexed and centrifuged. The benzene layer was recovered and separated on a GC system (Shimadzu GC2010) equipped with a Zebron ZB-WAX plus column (100 m, 0.25 mm ID, 0.25 m fi lm thickness). The oven temperature was programmed from 30°C (2 min hold) to 180°C at 20°C/min (2 min hold), then to 207°C at 4°C/min (3 min hold), then to 220°C at 2°C/min (2 min hold), and fi nally to 240°C at 2°C/min (2 min hold). Detection was performed with a fl ame ionization detector at 250°C to resolve fatty acids peaks, which were identifi ed using authentic standards (Restek Corp., Bellefonte, PA). Data was analyzed with Shimadzu's GC solutions software.
Fatty acid analysis of the phospholipid and triglyceride fractions was performed by extracting and preparing cell lysates as of the stearic acid-induced apoptosis and necrosis were eradicated.

Effect of oleic acid supplementation on stearic acid-induced activation of caspase-3
Caspase-3 is a key executioner enzyme associated with programmed cell death (i.e., apoptosis). Activation of caspase-3 requires proteolytic cleavage into fragments, which can target the nuclear enzyme poly ADP-ribose polymerase (PARP). Stearic acid supplementation (75 M) instigated an accumulation of cleaved caspase-3 within 24 h; however, this pro-apoptotic response was prevented with oleic acid cosupplementation ( Fig. 2 ). The counteractive effects of oleic acid cosupplementation were observed at the lowest concentration assessed. These data are consistent with the apoptosis and necrosis fi ndings in that cosupplementation of stearic acid with oleic acid (>25 M) eliminated the initiation and progression of programmed cell death. In parallel, these data support the fi ndings of oleic acid's ability to attenuate the stearic acid-induced cytotoxicity in HAECs.

Effect of oleic acid supplementation on stearic acid-induced ICAM-1 surface expression
Increased surface expression of ICAM-1 in endothelial cells is indicative of a pro-infl ammatory phenotype; therefore, we ascertained the effect of stearic acid in combination with oleic acid supplementation on the relative abundance of ICAM-1 expression. HAECs were treated with varying concentrations of oleic acid with or without stearic acid (50 M) for 24 h under standard culture conditions. Our investigation previously demonstrated that 50 M of stearic acid is growth inhibitory but not apoptotic or necrotic ( 17 ). Stearic acid supplementation at levels that do not induce cytotoxicity increased ICAM-1 surface expression on HAECs in a dose-dependent manner.   Before apoptosis/necrosis analysis using fl ow cytometry, endothelial cells were supplemented for 24 h with oleic and stearic acids, alone or in combination. The data refl ects the percentage of cells observed in each classifi cation. Results are expressed as the mean ± SD of three determinations. a Signifi cant differences ( P < 0.05) using Students' t -test compared to the vehicle control.
pression of ICAM-1 among many other proteins ( 44 ). Our previous investigation reported that stearic acid induced phosphorylation of I B-␣ and NF-B, the transcriptional regulators of ICAM-1 ( 17 ). HAECs supplemented with stearic acid (50 M) induced phosphorylation of the p65 subunit of NF-B ( Fig. 4 ). Oleic acid supplementation did not alter the baseline phosphorylation status; however, when stearic acid (50 M) was cosupplemented with oleic acid, the phosphorylation state of NF-B remained at or below baseline activation. The counteractive effects of oleic acid cosupplementation were observed at the lowest concentration assessed.

Cellular incorporation of stearic and oleic acid
HAECs were supplemented with an equivalent quantity of stearic and oleic acids alone and in combination for 24 h prior to fatty acid extraction and GC analysis. Table 2 summarizes the results of the targeted fatty acid analysis. Nonsupplemented vehicle cells consistently contain similar quantities of stearic and oleic acid in their composi-surface expression in HAECs. When HAECs were incubated with oleic acid in conjunction with stearic acid (50 M), the stearic acid-induced upregulation of ICAM-1 was completely suppressed. The inhibition of the stearic acid-induced increase in ICAM-1 expression was eliminated at even the lowest amount of oleic acid supplementation (5 M). Notably, ICAM-1 surface expression levels were not altered upon HAEC supplementation with oleic acid alone.

Effect of oleic acid supplementation on stearic acid-induced phosphorylation of NF-B
Stearic acid treatment signifi cantly increased cell surface expression levels of ICAM-1; subsequently, this proinfl ammatory response was prevented upon oleic acid cosupplementation ( Fig. 3 ). Activation of NF-kB by phosphorylation of the p65 subunit can lead to increased ex- Fig. 3. Effect of oleic acid on stearic acid-induced HAEC upregulation of ICAM-1. Endothelial cells were supplemented for 24 h with oleic and stearic acids, alone or in combination. Relative ICAM-1 membrane expression levels were quantifi ed by fl ow cytometry and presented as the mean fl uorescent intensity ± SD of three determinations. *Statistically signifi cant differences ( P < 0.05) from vehicle using Student's t -test. HAEC, human aortic endothelial cell; ICAM-1, intercellular adhesion molecule-1.

Fig. 2.
Effect of oleic acid on stearic acid-induced activation of caspase-3. HAECs were treated with stearic and oleic acids, alone and in combination, for 24 h at 37°C in 5% CO 2 . HAEC lysates (15 g each) were electrophoretically separated in a 4-12% polyacrylamide gradient gel and transferred onto a nitrocellulose membrane. The blot was probed with an antibody that recognizes the cleaved active form of caspase-3, which is present as doublet bands. GAPDH served as the protein loading control. This experiment was repeated to confi rm the appearance of cleaved caspase-3 in HAECs supplemented with stearic acid alone. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAEC, human aortic endothelial cell. Fig. 4. Effect of oleic acid on stearic acid-induced phosphorylation of NF-B, the transcriptional regulator of ICAM-1. A: HAECs were treated with stearic and oleic acids, alone and in combination at varying doses, for 24 h at 37°C in 5% CO 2 . HAEC lysates (15 g each) were electrophoretically separated in a 4-12% polyacrylamide gradient gel and transferred onto a nitrocellulose membrane. A phospho-specifi c antibody that recognizes the phosphorylation of the serine 536 residue on the p65 regulatory subunit of NF-B determined the relative activation state of the enzyme. A nonphospho-specifi c antibody to NF-B p65 and GAPDH served as protein loading controls. B: Quantifi cation of the phosphorylated NF-B p65 in relationship to the loading controls was determined by densitometry analysis. Data represent the mean values of three independent blots ± SD. *Statistically significant differences ( P < 0.05) from vehicle using Student's t -test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAEC, human aortic endothelial cell; ICAM-1, intercellular adhesion molecule-1; NF-B, nuclear factor-kappa B. HAECs were treated with oleic and stearic acids, alone or in combinations, for 24 h. Lipid extracts were analyzed by GC to determine the fatty acid profi les for each treatment Results were calculated based on the addition of an internal standard and normalized to protein content. Data represent the mean ± SD of three independent treatments as the percentage of fatty acid composition.

Phospholipid and triglyceride fatty acid characterization
Clearly, HAECs supplemented with stearic and oleic acids dramatically altered the fatty acid profi le; therefore, we set forth to determine where these fatty acids were incorporated. Phospholipid and triglyceride fractions were obtained from HAECs supplemented with either 50 M stearic acid, oleic acid, or a combination of both. The fatty acid composition of the phospholipid components are summarized in Table 4 . Stearic acid-supplemented cells signifi cantly increased their C18:0 content. A slight increase in C18:1n-9 was also observed, while palmitic acid assumed the greatest loss of any individual fatty acid. HAECs supplemented with 50 M oleic acid substantially increased the amount of oleic acid present in the phospholipid component. Moreover, the fatty acid combination resulted in a fatty acid pattern that was intermediate between the stearic acid-and oleic acid-supplemented cells.
The triglyceride component ( Table 5 ) was relatively small compared to the fatty acid content present in phospholipids ( Table 4 ). The amount and composition of the triglycerides in stearic acid-supplemented HAECs did not substantially differ from vehicle controls; however, a small increase in C18:0 was observed. HAECs supplemented with oleic acid generated a notable increase in the amount of fatty acids stored in the form of triglycerides, which predominantly consisted of oleic acid. Furthermore, the stearic and oleic acid combination resulted in an increase in triglyceride content but at levels that fell short of oleic acid supplementation alone. Oleic acid was a major contributor to the fatty acid triglyceride composition, followed by stearic and palmitic SFAs.

Accumulation of fatty acids in the form of triglycerides
To confi rm the triglyceride accumulation in endothelial cells supplemented with oleic acid, we utilized an Oil red O staining kit to visualize the lipid droplets. Fig. 6 represents HAECs supplemented with 50 M of oleic acid, stearic acid, or a combination of both compared to vehicle-treated cells. Excess triglycerides packaged in the form of lipid droplets were not readily apparent in vehicle and stearic acid-supplemented cells. However, an accumulation of lipid droplets were prominently displayed in endothelial cells supplemented with oleic acid, either in the presence or absence of stearic acid.

DISCUSSION
Parenteral and enteral nutrition (including oral diets) is utilized to provide healthy and ill patients with an energy source in the form of lipids. The lipids also provide essential fatty acids to these individuals. Commercially available lipids are derived from various oils that often vary between plant and animal origins. As a result, the fatty acid content of the lipids differs signifi cantly. SFAs represent a sizable component (15-55%) of the percentage of fatty acids present in most diets used for either parenteral or enteral administration. Our previous investigation evaluated the impact of individual tion. In HAECs supplemented with 25 M stearic acid, a notable increase in C18:0 was observed. Interestingly, these cells had a corresponding increase in C18:1n-9, which can be attributed to conversion from C18:0. Cells supplemented with 25 M oleic acid more than doubled the C18:1n-9 cellular content. This increase was largely at the expense of the SFAs, specifi cally C16:0 and C18:0, and arachidonic acid to a lesser extent. When both fatty acids were equally cosupplemented, analogous increases were observed, albeit the levels were considerably lower than either fatty acid supplemented individually.
On the basis of our previous experimental data, it is apparent that HAECs supplemented with greater than 50 M stearic acid initiate detrimental effects ( 17 ). We have shown in this study that these stearic acid-induced negative cellular effects can be essentially eliminated with oleic acid. Therefore, we investigated the impact of oleic acid upon stearic acid cellular incorporation. Table 3 summarizes the effect of oleic acid dose on the fatty acid profi le of endothelial cells supplemented with stearic acid (50 M). Stearic acid supplementation alone increased the overall cellular quantity of stearic acid (C18:0) to one-half of the total fatty acid content, which is nearly triple the amount present in control cells. Interestingly, cosupplementation of stearic acid with as little as 5 M oleic acid dramatically reduced the percentage of C18:0 in the cells ( Fig. 5 and Table 3 ). At concentrations greater than 10 M oleic acid, C18:0 was overtaken by C18:1n-9 in terms of percentage of the fatty acid composition, and the differences are exaggerated at even higher concentrations. When equimolar concentrations of both stearic and oleic acids are presented to endothelial cells, oleic acid is preferentially incorporated ( Table 3 ). Stearic acid-treated (50 M) HAECs were cosupplemented with varying doses of oleic acid (5-50 M) for 24 h. Albumin-treated cells served as the vehicle for a baseline comparison. Lipid extracts were analyzed by GC to determine the fatty acid profi les for each treatment. Results were calculated as described. Data represent the ± SD of three independent treatments as the percentage of fatty acid composition.
HAEC, human aortic endothelial cell.  Table 3 , a comparison of C18:0 and C18:1n-9 incorporation is presented as percentage of total fatty acid cellular content. Data represent a mean ± SD of three determinations. HAEC, human aortic endothelial cell.
SFAs upon endothelial cell fatty acid composition, growth, and functional responses ( 17 ). Long-chain SFAs, stearic acid in particular, evoked pro-infl ammatory responses and elevated quantities of stearic acid in the cells, resulting in endothelial cell apoptosis and necrosis ( 17 ). Long-chain SFAs are believed to be substantial contributors to atherosclerosis and coronary heart disease ( 1 ). Diets rich in oleic acid (i.e., Mediterranean diets) have been reported to reduce the risk of atherosclerotic disease, such as CAD ( 33 ). Therefore, we hypothesized that oleic acid antagonizes the effects of long-chain SFAs, such as stearic acid, on vascular cells. The purpose of this investigation was to determine the effects of oleic acid and stearic acid on endothelial cell integrity, function, and lipid incorporation. Our results indicate that oleic acid inhibits the toxic effects of stearic acid on endothelial cells in culture. Initially, our studies focused on the effect of oleic acid cosupplementation on stearic acid-induced growth inhibition and cytotoxicity. HAECs treated with stearic acid are growth-inhibited at lower concentrations (0-50 M); however, when exogenously added stearic acid surpassed 50 M, signifi cant endothelial cell loss from both apoptosis and necrosis was observed. In our study, both the growth inhibition and cell death induced by stearic acid was inhibited upon cosupplementation with oleic acid.
We substantiated these fi ndings using a fl ow cytometrybased apoptosis and necrosis detection assay. Stearic acidinduced apoptosis and necrosis was circumvented upon cosupplementation with 25 M oleic acid. SFAs are known to induce apoptosis in a variety of cell types, including, but not limited to, breast cancer cell lines, hematopoietic cell lines, and primary cell types ( 19,38,(45)(46)(47). Artwohl et al. published observations of stearic acid-induced apoptosis in HAECs and human umbilical vein endothelial cells (HUVEC) ( 20,46 ). In the current study, we report the generation of cleaved caspase-3 in cells treated with 75 M stearic acid. Consistent with our apoptosis and necrosis data, the appearance of cleaved caspase-3 was inhibited with oleic acid. Cleaved caspase-3 is well known to induce apoptosis ( 48 ). Endothelial cells were treated with stearic and oleic acids (50 M), alone or in combination, for 24 h before analysis. Triglycerides were acquired simultaneously with the phospholipid component as described. Fatty acid quantifi cation was based on an internal standard and normalized to protein content. Results are expressed as the mean ± SD of three determinations. "ND" indicates fatty acids that were not detected in the triglyceride profi le. ylation of the p65 subunit of NF-B. HAECs supplemented with oleic acid alone did not alter the baseline phosphorylation status of NF-B. However, cosupplementation of stearic with oleic acid diminished the activation of NF-B, which is consistent with the reduction in ICAM-1 surface expression levels.
To discern how these individual and combinations of fatty acids altered the fatty acid composition of cells, HAECs were treated with fatty acids for 24 h under standard tissue culture conditions, and GC assessment of the HAEC fatty acid content was determined. The per-Our laboratory previously reported the pro-infl ammatory effects of long-chain SFAs on endothelial cells ( 17 ). We reported that stearic acid-supplemented HAECs signifi cantly upregulated ICAM-1 surface expression when exposed to concentrations of stearic acid as low as 10 M. In the current study, cells treated with 50 M stearic acid increased ICAM-1 expression 2-fold. Furthermore, this statistically signifi cant increase in the pro-infl ammatory marker was diminished to basal levels with as little as 5 M oleic acid cosupplementation. Stearic acid-supplemented HAECs also demonstrated an increase in serine phosphor- Endothelial cells were treated with stearic and oleic acids (50 M), alone or in combination, for 24 h before analysis. Phospholipids were resolved using thin layer chromatography and extracted as described in Materials and Methods. Fatty acid quantifi cation was based on an internal standard and normalized to protein content. Results are expressed as the mean ± SD of three determinations. effects of oleic acid may be its ability to lower stearic acid levels in cells.
Listenberger et al. suggested that oleic acid prevents palmitic acid-induced apoptosis in Chinese hamster ovary cells by channeling palmitic acid into neutral lipids in the form of triglycerides ( 36 ). In our study, HAECs were treated with either stearic acid, oleic acid, or a combination of both; subsequently, the fatty acid profi les of the phospholipids and triglycerides were determined. Similar changes in the fatty acid profi le of the phospholipids were observed compared to the whole cell lipid extracts. Of particular interest, 50 M stearic acid treatment did not markedly increase the quantity of triglyceride cellular fatty acid; however, both oleic acid and the combination of stearic and oleic acids demonstrated an accumulation of triglycerides. This observation was confi rmed by staining fatty acid-supplemented endothelial cells with Oil Red O to visualize the cells' neutral lipid storage. In stark contrast to Listenberger's fi ndings, cosupplementation with stearic and oleic acids did not cause an increase in the quantity of C18:0 packaged into triglycerides ( 36 ). The Listenberger study suggests that the protective effect of oleic acid on palmitic acid-induced apoptosis is not a result of a decrease in palmitic acid uptake in the presence of oleic acid. In contrast, our results suggest that oleic acid diminishes uptake of stearic acid in endothelial cells.
In conclusion, our results are consistent with the hypothesis that the pro-infl ammation and cytotoxicity generated by an accumulation of stearic acid in cells can be diminished by relatively low amounts of oleic acid cosupplementation. Our evidence suggests that endothelial cells attempt to convert excessively incorporated stearic acid into less harmful oleic acid as a means of cell preservation. However, this process can lead to an abundance of C18:0 in the phospholipids, which can evolve into a pro-infl ammatory state. We hypothesize that too much C18:0 accumulates into the phospholipids, thereby creating an extremely rigid structure due to the tightly packed SFA content in the membrane, which results in lipotoxicity. These results indicate that longchain SFAs are potentially damaging to endothelial cells; however, if SFAs are presented to endothelial cells with at least a moderate quantity of oleic acid, the SFAinduced pro-infl ammatory and eventual cytotoxic effects can be averted. Our results provide mechanistic support to the benefi t of diets containing elevated levels of oleic acid (i.e., Mediterranean diet) on the development of infl ammatory and cardiovascular diseases. centage of C18:0 and C18:1n-9 fatty acids in the vehicletreated cells were remarkably similar; however, when HAECs were supplemented with either stearic or oleic fatty acids at equivalent concentrations, the fatty acid profi le was dramatically altered. The 25 M stearic acidtreated HAECs revealed an increase in the proportion of C18:0. Interestingly, there was a corresponding elevation in the proportion of C18:1n-9 in the stearic acidsupplemented HAECs, which was likely due to the conversion of C18:0 into C18:1n-9 by stearyol CoA desaturase. Incorporation of oleic acid resulted in a signifi cant increase in C18:1n-9, but this increase was primarily at the expense of stearic and palmitic acids and, to a lesser extent, arachidonic acid. When stearic and oleic acids were cosupplemented at equimolar concentrations, increases in both were readily apparent, albeit at more modest levels than in either one individually. The conversion of C18:0 into C18:1n-9 could be one mechanism by which endothelial cells resist the negative impact of incorporation of stearic acid. The results of the growth inhibition and ICAM-1 expression assays indicated that only a minimal quantity of oleic acid was required to offset the pro-infl ammatory and growth inhibitory effects induced by stearic acid. We treated HAECs with 50 M stearic acid in combination with varying amounts of oleic acid to determine alterations in the fatty acid profi le. Interestingly, cosupplementation of stearic acid with as little as 5 M of oleic acid dramatically decreased the percentage of C18:0 in the cells. Thus, one possible mechanism for the antagonistic Fig. 6. Effect of oleic and stearic acids on the accumulation of neutral lipid storage. HAECs were supplemented with 50 M of stearic acid, oleic acid, or both in combination for 24 h at 37°C in 5% CO 2 . As described in Materials and Methods, lipid droplets containing triglycerides were stained with Oil Red O, and nuclei were counterstained with hematoxylin for cellular defi nition. Only in cells supplemented with oleic acid, in the presence or absence of stearic acid, were lipid droplets readily visible as indicated by the arrows. No excess accumulation of lipid droplets was observed when HAECs were cosupplemented with both fatty acids. HAEC, human aortic endothelial cell.