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Journal of Lipid Research, Vol. 43, 944-951, June 2002
Copyright © 2002 by Lipid Research, Inc.

In vitro mimicry of essential fatty acid deficiency in human endothelial cells by TNF impact of -3 versus -6 fatty acids

Konstantin Mayer, Reinhold Schmidt, Marion Muhly-Reinholz, Tina Bögeholz, Stephanie Gokorsch, Friedrich Grimminger and Werner Seeger¹

Medizinische Klinik II, Zentrum für Innere Medizin, Justus-Liebig-University, Klinikstr. 36, D-35392 Giessen, Germany

¹ To whom correspondence should be addressed. e-mail: werner.seeger{at}innere.med.uni-giessen.de

	ABSTRACT

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Severe endothelial abnormalities are a prominent feature in sepsis with cytokines such as tumor necrosis factor (TNF) being implicated in the pathogenesis. As mimic to inflammation, human umbilical vascular endothelial cells (HUVEC) were incubated with TNF for 22 h, in the absence or presence of the -6 fatty acid (FA), arachidonic acid (AA), or the alternative -3 FA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). TNF caused marked alterations in the PUFA profile and long chain PUFA content of total phospholipids (PL) decreased. In contrast, there was a compensatory increase in mead acid [MA, 20:3(-9)], the hallmark acid of the essential fatty acid deficiency (EFAD) syndrome. Corresponding changes were noted in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, but not in the sphingomyelin fraction. Supplementation with AA, EPA, or DHA markedly increased the respective FA contents in the PL pools, suppressed the increase in MA, and resulted in a shift either toward further predominance of -6 or predominance of -3 FA.

We conclude that short-term TNF incubation of HUVEC causes an EFAD state hitherto only described for long-term malnutrition, and that endothelial cells are susceptible to differential influence by -3 versus -6 FA supplementation under these conditions.

Abbreviations: AA, 20:4-6, all-cis-5,8,11,14-eicosatetraenoic acid, arachidonic acid; CL, cardiolipin; DHA, 22:6-3, all-cis-4,7,10,13, 16,19-docosahexaenoic acid; EFAD, essential fatty acid deficiency syndrome; EPA, 20:5-3, all-cis-5,8,11,14,17-eicosapentaenoic acid; FA, fatty acids; HUVEC, human umbilical venous endothelial cells; LT, leukotriene; OA, 18:1-9, cis-9-octadecenoic acid, oleic acid; PAF, platelet-activating factor; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, prostaglandin; PI, phosphatidylinositol; PL, phospholipid; PS, phosphatidylserine; SPH, sphingomyelin; TNF, tumor necrosis factor; 14:0, tetradecanoic acid, myristic acid; 16:0, hexadecanoic acid, palmitic acid; 16:1-7, cis-9-hexadecenoic acid, palmitoleic acid; 18:0, octadecanoic acid, stearic acid; 18:2-6, all-cis-9,12-octadecadienoic acid, linoleic acid; 20:0, eicosanoic acid, arachidic acid; 20:1-9, cis-11-eicosenoic acid; 20:2-6, all-cis-11,14-eicosadienoic acid; 20:3-9, all-cis-5,8,11-eicosatrienoic acid, mead acid; 20:3-6, all-cis-8,11,14-eicosatrienoic acid; 20:3-3, all-cis-11,14,17-eicosatrienoic acid; 22:0, docosanoic acid, behenic acid; 22:1-9, cis-13-docosenoic acid, erucic acid; 22:4-6, all-cis-7,10,13,16-docosatetraenoic acid; 22:5-3, all-cis-7,10,13,16,19-docosapentaenoic acid; 24:0, tetracosanoic acid, lignoceric acid; 24:1-9, cis-15-tetracosenoic acid, nervonic acid

Supplementary key words arachidonic acid • eicosapentaenoic acid • mead acid • phosphatidylinositol • phosphatidylcholine

	INTRODUCTION

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In addition to cytokines, eicosanoids have long been implicated in inflammatory events occurring in sepsis (6, 10). Arachidonic acid (AA) is metabolized via multiple metabolic pathways including cyclooxygenase and various lipoxygenases to prostanoids, leukotrienes, and other lipoxygenase products (6, 10). Both pro- and anti-inflammatory functions have been ascribed to the various AA metabolites. The family of -6 fatty acids including AA represents the predominant PUFA in common Western diet. In contrast, -3 fatty acids make up an appreciable part of the fat in cold-water fish and seal meat. In this family of fatty acids (FA), the last double bond is located between the third and fourth carbon atom from the methyl end, with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) being prominent representatives. They serve as alternative lipid precursors for all metabolic pathways hitherto recognized for AA, with the formation of trienoic prostanoids (instead of the 2-series originating from AA) and 5-series leukotrienes (LT) (instead of the 4-series LTs derived from AA) (11). Interestingly, many of the -3 fatty acid-derived metabolites, including 5-series cysteinyl-LTs, LTB₅, and TxA₃, possess markedly reduced inflammatory and vasomotor potencies as compared with the AA-derived lipid mediators and may even exert antagonistic functions.

In addition to being precursors for different eicosanoids, -3 versus -6 fatty acid incorporation into membrane (phospho)-lipid pools was suggested to have impact on lipid-related intracellular signaling events (12–17). Phosphatidylinositol and sphingomyelin pools, but also subclasses of phosphatidylcholine such as the platelet-activating factor (PAF)-precursor pool, may be particularly relevant in this respect. This is of interest in view of the fact that diets with specific fat composition have attracted interest for modulating inflammatory and immunological processes (immunonutrition) (18–20). When offering -3 lipids such as fish oil via the enteral route it does, however, take several days to weeks to influence effectively the fatty acid composition of membrane (phospho)-lipids and thereby the lipid mediator profile in humans (21, 22). Pre-feeding animals with -3 lipids instead of -6 lipids was indeed found to enhance the survival in subsequently provoked septic events (23, 24). As an alternative approach, intravenous lipid emulsions, as used for parenteral nutrition under intensive care conditions, might be employed for rapid manipulation of the -3/-6 balance, by choosing either conventional preparations (-6 predominance) or a fish oil-based lipid emulsion (-3 predominance). High levels of the respective free fatty acids are readily detectable in septic patients undergoing parenteral nutrition with lipids (25). The lipid emulsions cause an activation of the endothelial lipoprotein lipase and the release of free fatty acids (26–28), which is greatly enhanced in septic patients where levels of free AA may surpass 100 µmol/l (25). The impact of these interventions on the fatty acid composition of endothelial (phospho)-lipid pools is, however, largely unknown.

Against this background, the current study employed human endothelial cells under TNF challenge as a mimic of systemic inflammatory conditions in vitro. We sought first putative changes in the fatty acid composition of relevant endothelial phospholipid pools in response to the cytokine challenge, and second for the impact -3 versus -6 fatty acid supplementation under these conditions. Most interestingly, both loss of PUFAs in the various phospholipid pools and enhanced appearance of the hallmark FA eicosatrienoic acid [20:3(-9), mead acid] indicated that 22 h of TNF incubation sufficed to provoke a severe essential fatty acid deficiency (EFAD) state in the endothelial cells, hitherto only recognized during long-term malnutrition (29) or under conditions of long-term lipid-deprived cell culture (30). When offered under these conditions, both -3 and -6 fatty acids were readily incorporated into the various phospholipid pools, with a corresponding shift of the -3/-6 balance. We speculate that the endothelial essential fatty acid homeostasis may be disturbed under conditions of severe inflammation and sepsis, which may have impact on lipid-dependent signaling pathways and mediator generation and is susceptible to differential influence by -3 versus -6 fatty acid supplementation.

	METHODS

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Cell culture
Primary cultures of human umbilical vein endothelial cells (HUVEC) were prepared by a modification of the method of Jaffe et al. (31) as previously described (32). For incorporation experiments, cells were washed and then incubated for 6 h in a 5% CO₂ atmosphere with MCDB 131 (Gibco, Karlsruhe, Germany) containing 1% FBS enriched with 10 µM fatty acids (free acids of [³H]AA, [³H]EPA, and [³H]DHA, Amersham Biosciences, Freiburg, Germany). Reactions were terminated by removal of the incubation medium and washing the cells twice with medium. For counting and extraction, HUVEC were harvested with trypsin (Gibco). Cells were washed and immediately frozen at -80°C under nitrogen.

Experimental protocol
In control experiments HUVEC were incubated in the absence of TNF and fatty acids in MCDB 131 supplemented with 1% FCS for 22 h. In all other groups, the incubation medium was supplemented with 10 ng/ml TNF (R & D, Wiesbaden, Germany) for 16 h after a pre-incubation of HUVEC for 6 h with either sham (+TNF-group) or 10 µmol/l of either AA, EPA, or DHA (free fatty acids from Sigma, Deisenhofen, Germany; TNF + AA, TNF + EPA, TNF + DHA groups, respectively). All experiments were performed in n = 6 independent cell cultures, unless otherwise stated.

Lipid analysis
Following treatment with ultrasound, cellular lipids were extracted using methanol-chloroform containing 0.01% butylated hydroxytoluene (BHT, m/v) according to the method of Bligh and Dyer (33). Total phospholipids (PL) were calculated using a colorimetric phosphorus assay (34). The distribution of PL classes was determined using high performance TLC as previously described (35). The one-dimensional system allows the separation of eight phospholipid classes: lyso-phosphatidylcholine (LPC), sphingomyelin (SPH), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidic acid (PA), and cardiolipin (CL). Staining was performed using molybdenum blue reagent according to Gustavsson (36). Quantification was done by means of densitometric scanning at 700 µm using a TLC-scanner II (Camag).

FA analysis of PL classes
Choline, serine, inositol, and ethanolamine phospholipids and sphingomyelin were separated on silica 60 thin layer plates (Merck, Darmstadt, Germany) with chloroform-methanol-acetic acid-water (50:37.5:3.5:2, v/v/v/v) as developing solvent. Following nondestructive visualization of individual and standard PLs with primuline (37), the corresponding gel compartments as well as blanks were eluted after addition of 10 µg pentadecanoic acid as an internal standard with 10 ml chloroform-methanol (v/v). The ester bound fatty acids were converted to fatty acid methyl ester (FAME) using acid-catalyzed transmethylation with 2 N HCl in methanol and analyzed using gas chromatography as described (38, 39).

Analysis of PC subclasses
Phosphatidylcholine subclasses were isolated by means of HPLC subsequent to phospholipolytic cleavage of the polar headgroup with phospholipase C and conversion of the resulting diradylglyceroles (DRG) with 1-naphthylisocyanate, with slight modifications of the method originally described by Ruestow et al. (40, 41). For fatty acid analysis, the alkyl-acyl subclass was subjected to gas chromatography as described (37).

Statistics
For statistical comparison, one-way ANOVA was performed. A level of P < 0.05 was considered to be significant.

	RESULTS

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Kinetics of free fatty acid uptake by endothelial cells
To determine the kinetics of free FA incorporation into endothelial cells, HUVEC were incubated with 10 µmol/l AA, EPA, or DHA, each spiked with radiolabeled FA. Within 2 h about 50–60%, and within 6 h about 80–90% of the label was found to be incorporated into the cells (data not given in detail).

Profile of phospholipid classes
Analysis of the phospholipid profile revealed no substantial differences between cells in the absence and presence of TNF challenge and those additionally incubated with the different PUFAs (Table 1). The most abundant PL was phosphatidylcholine (PC, 40%), followed by phosphatidylethanolamine (PE, 30%), phosphatidylserine (PS, 12%), and sphingomyelin (SPH, 9%). Phosphatidylinositol (PI, 5%) and cardiolipin (CL, 4%) appeared as minor compounds of total phospholipids. Other phospholipid classes (lyso-PC, phosphatidic acid) ranged below detection limits.

Fatty acid profile of phosphatidylcholine
The main fatty acids in the PC fraction of control HUVEC were palmitic acid (38%), oleic acid (30%), and stearic acid (9%) (Table 2). Compared with the fatty acids of total PL, the PC fraction contained relatively small amounts of long chain PUFA (3.6% AA, 0.6% EPA, and 1.7% DHA). Similar to total PL, TNF treatment of HUVEC resulted in an approximately 50% reduction of AA, EPA, and DHA, concomitant with an approximately 3-fold increase of 20:3(-9). In the presence of exogenous AA, the percentage of this fatty acid as well as 22:4(-6) was increased. Supplementation of HUVEC with EPA markedly augmented the EPA and the 22:5(-3) content in the TNF-exposed HUVEC, and supplementation with DHA was reflected by an increase of this fatty acid and of EPA in the PC fraction.

	DISCUSSION

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In the present study, incubation of human endothelial cells with TNF for 22 h caused marked alterations in the fatty acid profile of total phospholipids. Long chain PUFA content decreased, as evident for 18:2, 20:3(-6), 20:4, 20:5, 22:5(-3), and 22:6(-3), whereas mead acid [20:3(-9)] increased. Corresponding changes were noted in the phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol pools, but not in the sphingomyelin fraction. Supplementation with AA, EPA, or DHA under these conditions increased the respective fatty acid contents in the various phospholipid pools, with a corresponding shift in the -3/-6 ratio, and suppressed the increase in 20:3(-9).

In contrast to measurements in leukocytes (42, 43), only limited data on the fatty acid composition of the various phospholipid pools of human endothelial cells are currently available (44). Most impressively, 22 h incubation with TNF in a concentration range found in peak levels in septic shock patients (45) sufficed to provoke marked changes in the fatty acid composition of all, with the exception of sphingomyelin, endothelial phospholipid pools were investigated: a consistent decrease in long-chain PUFAs was noted. These changes correspond to the alterations in fatty acid composition encountered under conditions of essential fatty acid deficiency (EFAD) due to malnutrition (29, 30). It is in line with this view that the eicosatrienoic acid 20:3(-9), the hallmark acid of EFAD, was noted to be markedly increased in total phospholipids as well as in the PC, PE, PS, and PI pools. Of note, the 20:3(-9)/20:4 ratio, usually taken as parameter for quantifying the severity of EFAD (46), increased from 1:7 to 1:1.5 in PC and PI of endothelial cells under TNF challenge. Mead acid is normally present in animal tissue at very low quantities. It is the main PUFA synthesized by complete endogenous synthesis via elongation and desaturation of 18:1(-9), and its formation is upregulated upon lack of essential fatty acids. Mead acid itself is a poor substrate for elongation and desaturation and, due to the lack of a -6 double bound, for prostanoid and lipoxygenase product formation (46, 47).

Several mechanisms may underlie the depletion in PUFAs and the compensatory increase in mead acid in the TNF-exposed endothelial cells. In vitro growth of hepatic cells and HUVEC in delipidated medium was previously noted to mimic essential fatty acid deficiency with upregulation of 20:3(-9) within 10 days (30, 48). However, HUVEC were incubated in FCS supplemented medium with normal lipid contents for only 22 h, and control endothelial cells grown under these conditions displayed fully normal PUFA composition and mead acid-AA ratios. Furthermore, TNF may lead to activation of different phospholipolytic activities in various cell types, and preferential liberation of PUFAs, and in particular AA, was noted under these conditions (49). In addition to enhanced phospholipolytic activities, however, reduced re-incorporation of the PUFAs would be expected to result in such a markedly disturbed PUFA balance of the various phospholipid pools. In addition, TNF may proceed via increased oxygen radical attack on the sensitive unsaturated domains of PUFAs, whether membrane-incorporated or liberated by phospholipolytic activity, in the TNF-stimulated HUVEC. To the best of our knowledge, the quantitative relevance of such mechanisms in endothelial cells in the absence of neutrophil attack has hitherto not been addressed in detail (50–52); however, reduced intracellular glutathione levels were previously noted in endothelial cells undergoing TNF challenge (51, 52). Finally, endothelial cells possess limited ability to synthesize AA from the precursor linoleic acid due to low basal rates of elongases and desaturases (30, 53) adding to the loss of PUFA. Notably, the marked changes in HUVEC phospholipid FA composition in response to the presently used TNF concentration occurred in the absence of overt cell damage, as ascertained by lactate dehydrogenase release and trypan blue exclusion (data not given in detail).

Offering exogenous AA to the TNF-activated endothelial cells resulted in a marked increase in the percentage of this FA in the various phospholipid pools as compared with mono-cytokine challenge. Notably, due to near normalization of the AA content in the various phospholipid pools but still reduced EPA and DHA levels under TNF challenge, the (EPA + DHA)/AA ratio was markedly decreased under conditions of AA supplementation.

Supplementation of TNF activated HUVEC with EPA or DHA resulted in similarly impressive rates of incorporation of the respective fatty acids into the various phospholipid pools as noted for AA. Calculated for all phospholipids, the percentages of EPA and DHA in the TNF/EPA and TNF/DHA groups, respectively, were even manifold increased compared with control EC. This incorporation rate resulted in a dramatic change in the -3 (EPA + DHA)/-6 (AA) eicosanoid precursor fatty acid ratio in all phospholipid pools: in PI, the ratio increased from 1:9.4 to 1:2.8–1:2.4, and in the other PL pools the content of EPA plus DHA even surpassed the content of AA. Such an impressive shift of the -3/-6 ratio toward the alternative precursor fatty acids was hitherto only noted, for example, in leukocytes after long term dietary intake of fish oil capsules (17). In addition, the increase in 22:5(-3) in EPA-treated HUVEC and in 20:5(-3) in DHA-treated cells indicated some further conversion of these fatty acids in the endothelial cells. Notably, the increase in the EFAD indicator fatty acid 20:3(-9) was similarly suppressed under conditions of EPA or DHA supplementation as observed for supply with AA.

Of note, the concentration of AA, EPA, and DHA used in our experimental protocol is well in the range of these free fatty acids under oral supplementation with fish oil capsules and much lower as achieved under conditions of total parenteral nutrition of septic patients (25).

Although assessment of the functional consequences of the currently observed marked changes in fatty acid composition of the various endothelial phospholipid pools was beyond the scope of the present study, a marked impact of these changes on metabolic and signaling events is to be expected. The appearance of mead acid, representing a poor substrate for many metabolic pathways, in companion with decreased percentages of PUFAs, has been implicated in abnormalities in prostanoid formation and PI-dependent signaling pathways in the EFAD syndrome (54). The -3/-6 ratio in pools serving as precursors for eicosanoid formation, in particular the PI pool and the PC pool including the PAF-precursor subpool, will have an impact on prostanoid formation by the endothelial cells themselves (55).

In conclusion, this is the first report to describe that short-term incubation of human endothelial cells with TNF in doses not provoking cell death reproduce the criteria of essential fatty acid deficiency in vitro, i.e., loss of PUFAs in various phospholipid pools and upregulation of mead acid [20:3(-9)] as EFAD hallmark fatty acid. Supplementation with AA, EPA, or DHA under these conditions resulted in rapid incorporation into various phospholipid pools, reversed the increase in mead acid, and at the same time profoundly shifted the -3/-6 ratio of the endothelial cells to further predominance of -6 (AA) or to near equimolar presence of -3 and -6 (EPA, DHA). Both the cytokine-induced essential fatty acid deficiency and the susceptibility to -3- versus -6-fatty acid supplementation may be relevant for endothelial abnormalities and responses to parenteral nutrition with lipid emulsions in patients suffering from sepsis or SIRS.

	ACKNOWLEDGMENTS

 
This study was supported by the Deutsche Forschungsgemeinschaft (SFB 547 "Kardiopulmonales Gefäßsystem"). K.M. is recipient of a Novartis Research Scholarship. The authors thank PD Dr. Snipes for thorough linguistic editing of the manuscript. Portions of the doctoral thesis of T.B. are incorporated in this report.

Manuscript received October 15, 2001 and in revised form March 14, 2002.

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