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Institut für Ernährungswissenschaften, Martin-Luther-Universität Halle-Wittenberg, D-06108 Halle/Saale, Germany
2To whom correspondence should be addressed. E-mail: eder{at}landw.uni-halle.de.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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as a stable product of PGI2, thromboxane (TX) B2 as a stable product of TXA2, and PGE2] and NO in the medium were determined. Cells treated with 50 µmol/L of either cis-9, trans-11 or trans-10, cis-12 CLA released less of all of the eicosanoids and NO than control cells treated with medium alone (P < 0.05). The ratio between the amounts of 6-keto-PGF1 and that of TXB2 released did not differ between control cells and cells treated with either CLA isomer. Moreover, cells treated with 50 µmol/L of cis-9, trans-11 or trans-10, cis-12 CLA had a lower amount of arachidonic acid in their phosphatidylethanolamine fraction and a lower mRNA concentration and activity of secretory phospholipase A2 than control cells (P < 0.05). These data suggest that eicosanoid formation was impaired by a reduced availability of arachidonic acid for the cyclooxygenase pathway. In conclusion, this study shows that cis-9, trans 11-CLA and trans-10, cis-12 CLA influence the release of various eicosanoids and NO from human aortic endothelial cells. The effects observed in this study might be important because eicosanoids and NO released from endothelial cells are involved in the regulation of vessel tone and platelet aggregation. The results of the present study suggest that both CLA isomers had unfavorable effects on endothelial function.
KEY WORDS: • conjugated linoleic acids • endothelial cell • eicosanoids • nitric oxide
The endothelium plays an important role in the regulation of vasomotor tone, platelet aggregation and smooth muscle cell proliferation and migration. The main vasorelaxing factor produced by endothelial cells is nitric oxide (NO), which is synthesized by endothelium nitric oxide synthase (eNOS),2 a heme-containing oxygenase. NO formation can be activated by several factors such as physical stimuli, hormones, various cytokines or factors formed during blood coagulation such as thrombin (1). NO also inhibits aggregation of platelets and proliferation of smooth muscle cells. Prostacyclin (PGI2) and thromboxane A2 (TXA2), which are members of the eicosanoid family, are formed in endothelial cells from arachidonic acid. Arachidonic acid is a normal component of the phospholipids of endothelial cell membranes and derives mainly from 
5- and 6-desaturation of linoleic acid (2). It becomes available for eicosanoid synthesis only after it is released from phospholipid moieties by the action of phospholipase A2 (PLA2). Arachidonic acid released from phospholipids is converted to prostaglandin (PG) H2 by cyclooxygenases (COX-1 and COX-2). COX-1 is constitutively expressed, whereas the expression of COX-2 can be markedly enhanced upon cell activation such as in an inflammatory response (3). PGH2 is further enzymatically transformed to several prostaglandins such as prostaglandins D2, E2, F2, I2 and TXA2. Prostaglandins play an extraordinarily important role in vascular homeostasis. PGI2, through activation of the IP receptor present in smooth muscle cells and platelets, causes vasodilatation and inhibits platelet aggregation (4). TXA2, on the contrary, has vasoconstrictory and proaggregating effects (5). PGE2 is a much less potent vasodilator than PGI2. In view of their counteracting effects, the balance between PGI2 and TXA2 is important for the maintenance of vascular homeostasis. A decrease in this ratio is associated with pathophysiologic conditions such as thrombosis and ischemia (6,7).
It has been well established that the function of endothelial cells, particularly their production of eicosanoids, can be altered by modulating their membrane fatty acid composition, particularly the relative amounts of arachidonic acid and eicosapentaenoic acid (8). Several studies showed that conjugated linoleic acids (CLA), a group of positional and geometric isomers of linoleic acid characterized by the presence of conjugated double bonds, reduce the formation of eicosanoids in various animal cells and tissues (9–12). Recently, it was shown that CLA also inhibits the expression of inducible nitric oxide synthase (iNOS) and NO production in murine macrophages (13). Less information is available to date about the effects of CLA on the production of eicosanoids and NO in endothelial cells. The aim of this study was therefore to investigate the effects of two different isomers of CLA, cis-9, trans-11 CLA and trans-10, cis-12 CLA on the formation of vasoactive eicosanoids and NO in human aortic endothelial cells, which were chosen as a model. The cis-9, trans-11 CLA was considered because it is the predominant CLA isomer in milk and dairy products; >80% of CLA present in milk exists as the cis-9, trans-11 isomer (14). Trans-10, cis-12 CLA is present in milk and most other foods only in trace amounts, but it is of particular interest because it is more active than cis-9, trans-11 CLA in several respects (15,16). The mechanism by which CLA inhibits the formation of eicosanoids in various cell types is not yet fully understood. Some studies suggest that inhibition of eicosanoid formation by CLA is caused through a reduced arachidonic acid concentration in membrane phospholipids (7,10); others suggest that it is due to an inhibition of PGH synthase (17) or COX-2 (13,18,19). To investigate possible mechanisms involved in altered production of eicosanoids by CLA isomers in endothelial cells, we proposed to determine the amounts of arachidonic acid in endothelial phospholipids as well as gene expression or activities of enzymes involved in the formation of eicosanoids (5-, 6-desaturase, secretory PLA2, COX-1, COX-2). We studied gene expression of eNOS as the key enzyme in the production of NO.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cis-9, trans-11 CLA (
96% pure) and trans-10, cis-12 CLA (98% pure) isomers were obtained from Cayman Chemicals (Ann Arbor, MI). Endothelial cell basal medium MV (ECBM) with SupplementPack, Hepes buffered salt solution (HepesBSS), trypsin-EDTA (0.025% trypsin and 0.01% EDTA) and trypsin neutralizing solution (TNS) containing 0.05% trypsin inhibitor and 0.1% bovine serum albumin (BSA), were purchased from PromoCell (Heidelberg, Germany). HBSS and gentamicin were purchased from Invitrogen (Karlsruhe, Germany).
Cell culture.
Human aortic endothelial cells, obtained from a 38-y-old female donor, were purchased from PromoCell. The cells were isolated from the ascending aorta or the combination with aortic arch by enzymatic digestion. PromoCell characterized the cells by Factor VIII-related antigen expression. The cells were cultured in ECGM MV, composed of ECBM and supplements, including 5% fetal calf serum, 4 g/L endothelial cell growth supplement/heparin, 10 µg/L epidermal growth factor, 1 mg/L hydrocortisone, 50 µg/L amphotericin B and 50 mg/L gentamicin. Cells were passaged after reaching confluence by using trypsin/EDTA. After trypsinization, TNS was added to prevent enzymatic damage to the cells. Only cells from passages 3–10 were used for this study. After they reached 70–80% confluence, the cells were incubated for 24 h at 37°C with fresh medium alone (control), or with medium supplemented with either cis-9, trans-11 CLA or trans-10, cis-12 CLA at final concentrations of 5 or 50 µmol/L. At the end of the incubation periods, cells were rinsed with HepesBSS, harvested by trypsinization and pelleted by centrifugation (170 x g for 5 min). The cell pellets were washed twice with HBSS, resuspended in an aliquot of HBSS and incubated for 1 h at 37°C.
Cell count, viability and protein determination.
The cell count was determined with a Neubauer chamber. Cell viability was examined by the trypan blue dye exclusion method. Protein concentration was measured by the method of Bradford (20) with BSA (Sigma-Aldrich Chemicals, Deisenhofen, Germany) as standard.
Fatty acid solution.
Stock solutions of conjugated linoleic acids (cis-9, trans-11 CLA and trans-10, cis-12 CLA) were prepared in ethanol at a concentration of 100 mmol/L. For the preparation of the test media, aliquots of these stock solutions were used. The solvent was evaporated under nitrogen and the fatty acids were converted to their sodium salts by adding equimolar amounts of sodium hydroxide solution. The solution containing the fatty acid salts was added to the growth medium to obtain the final concentrations.
Lipid analysis.
The total cellular lipids were extracted with 1 mL hexane/isopropanol (3:2 v/v). The lipid extracts were dried under nitrogen. Individual lipid fractions of the extracts were separated by a solid-phase extraction method as described by Suzuki et al. (21) with modifications. Lipids were transmethylated with trimethylsulfonium hydroxide (22). FAME were separated by GC (23).
Release of eicosanoids from human aortic endothelial cells.
After the 24-h incubation with CLA isomers and the 1-h incubation in HBSS, cells were centrifuged (2400 x g for 3 min) and the supernatants were assayed for the eicosanoids 6-keto PGF1, PGE2 and TXB2 using EIA-kits (No. 515211, 514010 and 519031, Cayman Chemicals). 6-keto PGF1 and TXB2 were determined as measures of the unstable PGI2 and TXA2.
Activity of secretory PLA2 (sPLA2) in human aortic endothelial cells.
The sPLA2 activity was determined in the cell incubation medium after 24 h of treatment with the CLA isomers and in the supernatant after an additional 1-h incubation in HBSS by a sPLA2 Assay Kit (No. 765001, Cayman Chemicals).
Release of NO from human aortic endothelial cells.
As an index of the NO concentration, nitrate and nitrite concentrations were determined in the cell incubation medium after 24 h of treatment with CLA isomers and in the supernatant after the additional 1-h incubation in HBSS by a Nitrate/Nitrite Colorimetric Assay Kit (No. 780001, Cayman Chemicals).
mRNA expression of the enzymes 5-desaturase, 6-desaturase, secretory PLA2, COX-1 and COX-2, eNOS.
mRNA expressions were determined in the cells after 24 h of treatment with CLA isomers and the 1-h HBSS incubation. Total RNA of the cells was isolated using the Trizol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s protocol; 1.2 µg total RNA was used for the cDNA synthesis at 42°C for 1 h. Cell mRNA expressions of 5-desaturase, 6-desaturase, eNOS and COX-1 were estimated by quantitative real-time RT-PCR using a MJ Research Opticon system (Biozym Diagnostik GmbH, Oldendorf, Germany). cDNA was amplified in a 15-µL reaction containing 2 µL RT-mixture, Brilliant SYBRGreen QPCR Master Mix (Stratagene, Amsterdam, The Netherlands) and specific primers (Table 1). The primers for 6-desaturase, 5-desaturase, COX-1 and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized by Qiagen (Hilden, Germany) and the primer for eNOS was synthesized by Metabion (Martinsried, Germany). After an initial denaturation step (10 min, 95°C), PCR was carried out for 30–35 cycles. Each cycle comprised denaturation for 30 s at 95°C, annealing at primer-specific temperature (Table 1), elongation for 1 min at 72°C and a final extension for 7 min at 72°C. Cell mRNA expression of group V sPLA2 was estimated by real-time RT-PCR using the Rotor Gene 2000 system (Corbett Research, Mortlake, Australia). cDNA was amplified in a 15-µL reaction containing 2 µL RT-mixture, 5 U of Taq DNA polymerase (Promega, Mannheim, Germany), 0.375 µL 10X SYBRGreen I (Sigma) and group V sPLA2-specific primers obtained from Carl Roth (Karlsruhe, Germany). After an initial denaturation step (120 s, 95°C), PCR was carried out for 40–50 cycles, each cycle comprising denaturation for 20 s at 95°C, annealing for 30 s at 59°C and elongation for 40 s at 72°C, followed by a final extension for 7 min at 72°C. Fluorescence intensity in all real-time PCR probes was measured at the end of the extension step and related to GAPDH.
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Statistical analysis.
Treatment effects were analyzed using one-way ANOVA. For significant F-values, means were compared by Fisher’s multiple range test. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The growth and viability of the cells were not affected by incubation with either CLA isomer. Cell count after incubation with medium alone was 2.45 ± 0.47 x 106/flask; after incubation with 5 and 50 µmol/L of cis-9, trans-11 CLA and 5 and 50 µmol/L of trans-10, cis-12 CLA, cell counts were 2.44 ± 0.24, 2.85 ± 0.49, 2.55 ± 0.39 and 2.48 ± 0.35 x 106/flask (means ± SD, n = 6), respectively. Cell viability was 96 ± 3% for control cells, 95 ± 1 and 98 ± 1% for cells treated with 5 and 50 µmol/L of cis-9, trans-11 CLA and 95 ± 2 and 97 ± 2% for cells treated with 5 and 50 µmol/L of trans-10, cis-12 CLA (n = 6). The protein levels also did not differ in control cells (0.50 ± 0.14 mg/106 cells), cells treated with 5 and 50 µmol/L of cis-9, trans-11 CLA (0.48 ± 0.11 and 0.55 ± 0.20 mg/106 cells, respectively) and cells treated with 5 and 50 µmol/L of trans-10, cis-12 CLA (0.52 ± 0.14 and 0.63 ± 0.21 mg/106 cells, respectively) (n = 6).
Incorporation of CLA isomers into human aortic endothelial cell lipids.
The incorporation of both CLA isomers into endothelial cell lipids was dependent upon their concentrations in the medium (Table 2). Concentrations of either CLA isomer in endothelial cell lipids were much higher after incubation with 50 µmol/L of either CLA isomer than after incubation with 5 µmol/L. After incubation of cells with 5 µmol/L of both CLA isomers, the concentrations of either CLA isomer were similar in neutral lipids, PC and PE; after incubation with 50 µmol/L of both CLA isomers, the concentrations of either CLA isomer were significantly higher in PC than in PE or neutral lipids.
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The concentration of arachidonic acid was generally much higher in PE than in PC (Fig. 1). Both CLA isomers reduced the concentration of arachidonic acid in PE in a concentration-dependent manner; the effect was similar for both CLA isomers. The concentration of arachidonic acid in PC was not altered by treating cells with 5 or 50 µmol/L of either cis-9, trans-11 CLA or trans-10, cis-12 CLA.
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5- and 6-desaturase.
Relative mRNA concentration of 5-desaturase did not differ between control cells and cells treated with 5 or 50 µmol/L of cis-9, trans-11 CLA or trans-10, cis-12 CLA (data not shown). Cells treated with 50 µmol/L of trans-10, cis-12 CLA had a significantly lower relative mRNA concentration of 6-desaturase than control cells; cells treated with 50 µmol/L of cis-9, trans-11 CLA tended (P < 0.10) to have a lower mRNA concentration of that enzyme than control cells (Fig. 2). Cells treated with 5 µmol/L of cis-9, trans-11 CLA or trans-10, cis-12 CLA did not differ from control cells in their relative mRNA of 6-desaturase.
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, TXB2 and PGE2 from endothelial cells.
Cells treated with 5 µmol/L of cis-9, trans-11 CLA did not differ in the release of 6-keto-PGF1, TXB2 and PGE2 from control cells (Figs. 3and 4). Cells treated with 5 µmol/L of trans-10, cis-12 CLA released less 6-keto-PGF1 into the medium than control cells but did not differ from control cells in the release of TXB2 and PGE2 into the medium. Cells treated with 50 µmol/L of either cis-9, trans-11 CLA or trans-10, cis-12 CLA released less of all of the eicosanoids than control cells. Cells treated with 50 µmol/L of cis-9, trans-11 CLA did not differ from those treated with 50 µmol/L of trans-10, cis-12 CLA in the release of 6-keto-PGF1, TXB2 and PGE2. The ratio between the amount of 6-keto-PGF1 and that of TXB2 released did not differ between control cells (0.92 ± 0.40), cells treated with 5 and 50 µmol/L of cis-9, trans-11 CLA (0.82 ± 0.30 and 1.05 ± 0.51, respectively) and cells treated with 5 and 50 µmol/L of trans-10, cis-12 CLA (0.60 ± 0.35 and 0.86 ± 0.11) (means ± SD, n = 6).
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Cells treated with 5 µmol/L of either cis-9, trans-11 CLA or trans-10, cis-12 CLA did not differ from control cells in the relative mRNA concentration of sPLA2, whereas cells treated with 50 µmol/L of either cis-9, trans-11 CLA or trans-10, cis-12 CLA had a lower relative mRNA concentration of that enzyme than control cells (Fig. 5A). Cells that were treated for 24 h with 5 or 50 µmol/L of cis-9, trans-11 CLA had a significantly lower activity of sPLA2 than control cells; cells treated with 5 µmol/L of trans-10, cis-12 CLA did not differ from control cells in the activity of sPLA2; cells treated with 50 µmol/L of trans-10, cis-12 CLA tended to have lower activity of sPLA2 (P < 0.10) compared with control cells (Fig. 5B).
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Cells treated with 5 µmol/L of cis-9, trans-11 CLA had a higher relative mRNA concentration of COX-1 than control cells; cells treated with 50 µmol/L of cis-9, trans-11 CLA, 5 or 50 µmol/L of trans-10, cis-12 CLA did not differ from control cells in the relative mRNA concentration of that enzyme (Fig. 6). Cells treated with 50 µmol/L of cis-9, trans-11 CLA had a lower mRNA concentration of COX-2 than control cells; in cells treated with 50 µmol/L of trans-10, cis-12 CLA, the mRNA concentration of that enzyme tended to be lower (P < 0.10) than in control cells. Cells treated with 5 µmol/L of cis-9, trans-11 CLA or trans-10, cis-12 CLA did not differ from control cells in the relative mRNA concentration of COX-2.
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Endothelial cells treated with 5 or 50 µmol/L of cis-9, trans-11 CLA released less NO during a 1-h incubation in HBSS than control cells (Fig. 7). Cells treated with 50 µmol/L of trans-10, cis-12 CLA also released less NO than control cells; the release of NO did not differ between cells treated with 5 µmol/L of trans-10, cis-12 CLA and control cells.
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Relative mRNA concentration of eNOS did not differ between control cells and cells treated with 5 or 50 µmol/L of cis-9, trans-11 or trans-10, cis-12 CLA (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our study showed that treating endothelial cells with high concentrations of cis-9, trans-11 CLA or trans-10, cis-12 CLA reduced the release of PGI2, PGE2 and TXA2. Two other recent studies also showed that treatment of endothelial cells with CLA altered the release of various eicosanoids in human saphenous vein endothelial cells (25,26). In one study (25), both isomers reduced the release of PGI2 in thrombin-activated cells, whereas they stimulated the release of PGI2 in resting cells, which is contrary to our study. This shows that the effects of CLA may be different for resting and activated cells. In the other study (26), treatment with 50 µmol/L of either cis-9, trans-11 or trans-10, cis-12 CLA reduced the formation of various eicosanoids in resting or activated human endothelial cells, such as in our study, which was performed with resting cells.
The results of our study suggest that reduced formation of eicosanoids by CLA might be the result of a diminished availability of arachidonic acid, which is rate limiting in the production of eicosanoids. Reduced activity of sPLA2 together with a lower concentration of arachidonic acid in PE, the major substrate of PLA2, might reduce the concentration of arachidonic acid available for cyclooxygenase reaction. Lower concentrations of arachidonic acid in PE of endothelial cells treated with the CLA isomers might have been caused by reduced activity of 6-desaturase, the rate-limiting enzyme in the formation of arachidonic acid from linoleic acid. For technical reasons, we were not able to determine the activity of that enzyme; however, we assume that the reduced gene expression in endothelial cells treated with CLA was associated with reduced activity of that enzyme. Previous studies in cells and animals also showed that various CLA isomers suppress 6-desaturation of linoleic acid, leading to lower concentrations of arachidonic acid in cell or tissue lipids (16,27,28). The finding that trans-10, cis-12 CLA caused a stronger inhibition of 6-desaturase gene expression than cis-9, trans-11 CLA, whereas the concentrations of arachidonic acid in PE did not differ after treatment with both CLA isomers, is unexplained.
The application of semiquantitative RT-PCR showed that high concentrations of cis-9, trans-11 CLA reduced gene expression of the inducible enzyme COX-2, and high concentrations of trans-10, cis-12 tended to do so. Reduced gene expression of that enzyme by various CLA isomers was also observed in recent studies in murine macrophages and hepatoma cells (18,19). It was suggested that decreased gene expression of COX-2 in macrophages treated with CLA is caused by activation of peroxisome proliferator-activated receptor 
(19). Some studies found a correlation between the expression of COX and the formation of eicosanoids in cells treated with CLA (13,18,19). Our study showed that cis-9, trans-11 and trans-10, cis-12 CLA do not suppress gene expression of COX-1. However, the possibility exists that CLA or elongated and desaturated products from CLA such as conjugated eicosatetraenoate act as antagonists for COX, thereby reducing available enzyme for arachidonic acid (29).
Our study is the first to demonstrate that treatment of endothelial cells with cis-9, trans-11 or trans-10, cis-12 CLA lowers the release of NO. To determine possible reasons for this, we examined gene expression of eNOS, the principal enzyme of NO synthesis. Because gene expression of eNOS was not influenced by cis-9, trans-11 or trans-10, cis-12 CLA, reduced release of NO could be due either to reduced activity of that enzyme or to reduced activity of iNOS. Activation of eNOS requires several cofactors for maximum activity such as NADPH, 5,6,7,8-tetrahydrobiopterin, FMN, and FAD. Signal transduction processes that can lead to calcium-independent activation of eNOS involve tyrosine phosphorylation, activation of phospholipase C and an increase in 1,4,5-triphosphate (1). Expression of iNOS was not determined in this study. But in another study, expression of iNOS in murine macrophages was reduced by CLA (19).
To assess the biologic importance of the CLA treatment on endothelial cells, the effects and biological activities of the individual eicosanoids and NO must be considered. Both PGI2 and TXA2 have strong effects on platelet aggregation and vessel tone. Because they counteract each other in these effects, the ratio between them is more relevant with respect to aggregation and vessel tone than are their absolute concentrations (6). Because this ratio did not change, the effect of the CLA isomers in this respect might be neutral. But reduced concentrations of NO, a strong vasodilator and inhibitor of aggregation, and PGE2, a weak vasodilator, suggest that the total effect of CLA in endothelial cells favors platelet aggregation and vasoconstriction. These are unfavorable effects. However, we are aware that platelet aggregation and vessel tone are also strongly influenced by the function of other cells such as platelets or smooth muscle cells. It was shown that incubation of platelets with either cis-9, trans-11 or trans-10, cis-12 CLA inhibited the formation of TXA2 and aggregation (30). Therefore, CLA might influence aggregation and vessel tone also by its effect on platelets.
Because the effects in endothelial cells occurred at CLA concentrations that are higher than those expected in human cells in vivo, it is questionable whether the effects of the CLA isomers on the release of eicosanoids and NO are physiologically relevant. Because the effects of CLA on endothelial cells in vivo are also more complex than those observed in vitro, dietary studies with animals are required to determine whether the effects of CLA in the present study on the formation of eicosanoids and NO are relevant in vivo.
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FOOTNOTES |
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3 Abbreviations used: BSA, bovine serum albumin; CLA, conjugated linoleic acid; COX, cyclooxygenase; ECBM, endothelial cell basal medium; eNOS, endothelial nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HepesBSS, Hepes buffered salt solution; iNOS, inducible nitric oxide synthase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, prostaglandin; PGI2, prostacyclin; sPLA2, secretory phospholipase A2; TNS, trypsin neutralizing solution; TX, thromboxane.
Manuscript received 14 August 2003. Initial review completed 8 September 2003. Revision accepted 18 September 2003.
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