Antiinflammatory Effects of Tetradecylthioacetic AcidInvolve Both Peroxisome Proliferator–Activated Receptor

�–Dependent and –Independent PathwaysEndre Dyrøy, Arne Yndestad, Thor Ueland, Bente Halvorsen, Jan Kristian Damås,

Pål Aukrust, Rolf K. Berge

Objective—Tetradecylthioacetic acid (TTA) is a hypolipidemic antioxidant with immunomodulating properties involvingactivation of peroxisome proliferator–activated receptors (PPARs). Human endothelial cells express PPARs. Wehypothesized that TTA could modulate endothelial cell activation at least partly through PPAR-related mechanisms.

Methods and Results—We explored this hypothesis by different experimental approaches involving both in vitro studiesin human endothelial cells (HUVECs) and in vivo studies in humans and PPAR-��/� mice. Our main findings were asfollows: (1) TTA suppressed the tumor necrosis factor �–induced expression of vascular cell adhesion molecule 1(VCAM-1) and interleukin 8 (IL-8) in HUVECs. (2) No TTA-mediated attenuation of VCAM-1 and chemokineexpression was seen in the liver of PPAR-��/� mice. (3) Whereas TTA markedly enhanced PPAR-�–target genes in theliver of wild-type, but not of PPAR-��/�, mice, no such effect on PPAR-�–target genes was seen in HUVECs. (4) Therelevance of our findings to human disease was suggested by a TTA-mediated downregulation of serum levels of solubleVCAM-1 and IL-8 in psoriasis patients.

Conclusion—We show that TTA has the ability to attenuate tumor necrosis factor �–mediated endothelial cell activation,further supporting antiinflammatory effects of this fatty acid, possibly involving both PPAR-�–dependent and–independent pathways. (Arterioscler Thromb Vasc Biol. 2005;25:1364-1369.)

Key Words: endothelial dysfunction � inflammation � cytokine � peroxisome proliferator–activated receptors

Enhanced activation of the endothelium seems to play apathogenic role in several inflammatory disorders such

as atherosclerosis, diabetes, and various autoimmune disor-ders.1–3 Endothelial cell activation refers to changes in theendothelium as a result of, for example, cytokine stimulationduring inflammatory and infectious conditions.4,5 Typically,these changes include expression of adhesion molecules, suchas vascular cell adhesion molecule 1 (VCAM-1),6 and che-mokines, such as monocyte chemoattractant protein 1(MCP-1) and interleukin 8 (IL-8).7–9 The end result isrecruitment of leukocytes with transmigration of cells into thearterial wall promoting local inflammation and further re-cruitment of activated leukocytes, representing a pathogenicloop in several inflammatory disorders.10 Accordingly, endo-thelial cell activation represents an interesting pharmaceuticaltarget for prevention and modulation of various inflammatoryand cardiovascular diseases.11,12

3-Thia fatty acids such as tetradecylthioacetic acid (TTA),are modified fatty acids that promote hepatic proliferation ofmitochondria and peroxisomes and decrease serum triacyl-

glycerol, cholesterol, and free fatty acid levels in animalmodels.13–15 The chemical properties of TTA are similar tonormal fatty acids of similar length, but the metabolism andmetabolic effects of TTA differ markedly from these otherfatty acids.16,17 Whereas normal �-oxidation of TTA does notoccur, it is catabolized through �-oxidation and sulfur oxi-dation, and short dicarboxylic metabolites can be found in theurine of animals and patients receiving TTA. TTA can also bedesaturated, and a small proportion of its �9-desaturatedmetabolite is found in plasma and tissues of patients andanimals treated with TTA.

The hypolipidemic response after TTA treatment seems toinvolve activation of peroxisome proliferator–activated re-ceptors (PPARs) in the liver.18,19 However, it has been shownthat TTA is a ligand for all PPAR subtypes,20,21 indicatingthat TTA could also act through PPARs outside the liver.Moreover, TTA has been shown to have antioxidant effects invitro,22 and we demonstrated recently that TTA also hasimmunomodulatory properties in human peripheral bloodmononuclear cells (PBMCs)23 and in HIV-infected patients.24

Original received November 16, 2004; final version accepted May 11, 2005.From the Institute of Medicine (E.D., R.K.B.), Section of Medical Biochemistry, University of Bergen, Haukeland University Hospital, Bergen; and

the Research Institute for Internal Medicine (A.Y., T.U., B.H., J.K.D., P.A.), Section of Endocrinology (T.U.), and Section of Clinical Immunology andInfectious Diseases (P.A.), Medical Department, Rikshospitalet, University of Oslo, Oslo, Norway.

Correspondence to Endre Dyrøy, Section of Medical Biochemistry, Institute of Medicine University of Bergen, Haukeland University Hospital N-5021,Bergen, Norway. E-mail endre.dyroy@med.uib.no

© 2005 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000171982.57713.96

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However, it is not known whether TTA can modulateendothelial cell activation.

Human endothelial cells express both PPAR-� and PPAR-�,25,26 and activation of PPAR-�, but not PPAR-�,25 inhibitscytokine-induced VCAM-1 expression in human endothelialcells. We hypothesized that TTA could modulate endothelialcell activation and inflammation at least partly involvingPPAR-related mechanisms. In the present study, this hypoth-esis was explored by different experimental approachesinvolving in vitro studies in human endothelial cells, in vivostudies in psoriasis patients, and PPAR-��/� mice.

MethodsAn online Methods section is available in the data supplement athttp://atvb.ahajournals.org.

Preparation of Fatty AcidsTTA was prepared as previously described.27 Penn Pharmaceuticals(Gwent, UK) manufactured the capsules used in the psoriasis study.

Cell ExperimentsPBMCs and neutrophils were obtained from heparinized blood byIsopaque-Ficoll (PBMCs: Lymphoprep, Nycomed, Oslo, Norway;neutrophils: Polymorphrep, Axis Shieldh, Oslo) gradient centrifuga-tion. Human umbilical vein endothelial cells (HUVECs) werepurchased from PromoCell (C-12250; Heidelberg, Germany). Cellproliferation was assessed by [3H]thymidine incorporation.

Quantitative Real-Time ReverseTranscription–Polymerase Chain ReactionTotal RNA was isolated from HUVECs, mouse liver, and PBMCsusing RNeasy Minikit (Qiagen, Hilden, Germany) and reversed-transcribed using a reverse transcriptase kit (Applied Biosystems,Foster City, Calif). Quantification of mRNA was performed usingthe ABI Prism7000 (Applied Biosystems).29

Enzyme ImmunoassaysIL-8, MCP-1, soluble VCAM-1 (sVCAM-1), and tumor necrosisfactor � (TNF-�) protein levels were measured by enzyme immu-noassay (R&D Systems, Minneapolis).

Human Psoriasis PatientsA total of 43 patients (17 females and 26 males; all white; mean age,41.4 years [range, 22 to 66 years]) with psoriasis were randomized toplacebo or TTA (1 g/d) for 21 days in a double-blind fashion. Therewere similar key demographic and clinical signs of psoriasis in thetwo treatment groups, and the majority of the patients were assessedto have psoriasis of moderate severity (data not shown). The localethics committee and the Norwegian Medicines Agency approvedthe study. Signed informed consent was obtained from each patient.

Animal StudiesThe animal study has been previously described.30 Briefly, wild-typeand PPAR-��/� mice (20 to 25 g) were pure-bred on a SV129background.31 The Norwegian State Board of Biological Experi-ments with Living Animals approved the protocol.

StatisticsEach HUVEC experiment was performed in duplicate or triplicate(growth experiments with 8 parallels), and each experiment wasrepeated at least three times. Differences between groups were testedby Student t test. For comparison of human data, the Mann–WhitneyU test was used. Probability values (2-sided) were consideredsignificant at P�0.05.

ResultsTNF-� Activates Endothelial Cells in a Dose- andTime-Dependent MannerTNF-� markedly enhanced the expression of VCAM-1, IL-8,and endothelial (E-selectin) in HUVECs in a dose-dependentmanner, reaching a plateau at 1 ng/mL for IL-8 and 5 ng/mLfor VCAM-1 and E-Selectin (Figure IA, available online athttp://atvb.ahajournals.org). Time-course experimentsshowed a marked TNF-�–induced expression of these genesalready after 3 hours (Figure IB). However, whereas IL-8 andVCAM-1 mRNA levels continued to rise during the obser-vation period, reaching maximum after 20 hours, E-selectinexpression declined after 3 hours of TNF-� exposure (FigureIB).

TTA Reduces TNF-�–InducedEndothelial ActivationPretreatment with TTA attenuated the TNF-�–inducedmRNA expression of VCAM-1 and IL-8 but not of

Figure 1. A, Effect of different concentrations of TTA (pretreatment for 72 hours) on the gene expression of E-selectin, IL-8, andVCAM-1 in TNF-�–stimulated HUVECs after culturing for 6 hours. Data are given as mean�SEM of 3 experiments. mRNA levels werequantified by real-time reverse transcription–polymerase chain reaction (RT-PCR) and are presented relative to the gene expression ofthe house-keeping gene �-actin. B and C, Effect of TTA (10 �mol/L) on the release of IL-8 and sVCAM-1, respectively, in TNF-�–stimu-lated (10 ng/mL) HUVEC supernatants after culturing for 20 hours as assessed by enzyme immunoassay. Data are given asmean�SEM of 3 experiments. D, Effect of TTA (10 �mol/L) on the adhesion of monocytes and neutrophils to TNF-�–activated (10ng/mL) HUVECs. Data are given as mean�SEM of 6 experiments. *P�0.05 vs TNF-�–stimulated cells for all panels. Unstim indicatesunstimulated.

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E-selectin, with suppressive effects of TTA at concentrations�10 �mol/L (Figure 1A). A similar pattern was also seen atthe protein level in HUVEC supernatants, showing a suppres-sive effect of TTA (10 �mol/L) on the TNF-�–inducedrelease of sVCAM-1 and, in particular, of IL-8 (Figure 1Band 1C). In fact, TTA nearly abolished completely theTNF-�–induced release of IL-8. TTA has previously beenshown to inhibit cell growth and induce apoptosis in cancercells21,32,33 and smooth muscle cells.34 However, TTA did notreduce growth of endothelial cells at dosages up to100 �mol/L, as assessed by [3H]thymidine incorporation andhad no effect on total protein synthesis or mRNA concentra-tions (data not shown).

Effect of TTA on TNF-�–Induced Adhesion ofMonocytes and Neutrophils to HUVECsIt has previously been reported that certain PPAR agonistsmay help limit chronic inflammation mediated by VCAM-1and monocytes, without affecting acute inflammation medi-ated by E-selectin and neutrophil binding.35 Based on theability of TTA to downregulate VCAM-1, but not E-selectin,in HUVECs, we examined whether TTA modulated differ-ently the adhesion of neutrophils and monocytes to TNF-�–activated HUVECs. As shown in Figure 1D, whereas TTAsignificantly attenuated monocyte adhesion to TNF-�–acti-vated HUVECs, no such effect was seen on neutrophiladhesion.

TTA Increases Both PPAR-� and PPAR-� GeneExpression in Endothelial CellsTo examine whether PPARs were involved in the TTA-mediated suppression of VCAM-1 and IL-8 in HUVECs, we

measured the effect of TTA on the gene expression of PPARsin these cells. Notably, we found that TTA treatment en-hanced mRNA level of both PPAR-� (�2.5 fold) andPPAR-� (�1.7-fold) in TNF-�–treated HUVECs (Figure 2).The PPAR-� gene expression was too weak to yield reliablequantitative results.

TTA Treatment Does Not Attenuate VCAM-1,IL-8, or MCP-1 Expression in PPAR-��/� MiceTo further elucidate the role of PPARs in the antiinflamma-tory effects of TTA, we examined the ability of TTA tomodify the expression of VCAM-1, IL-8, and MCP-1 in theliver from wild-type and PPAR-��/� mice. As shown inFigure 3, TTA suppressed both VCAM-1 and, in particular,IL-8 and MCP-1 expression in the liver of wild-type mice. Incontrast, the opposite effect was seen in PPAR-��/� micewith an enhancing effect of TTA on these mediators (Figure3). The reason for this latter finding is not clear but mayreflect that some PPAR-�–independent enhancing effects ofTTA on these mediators are counter-acted by TTA-mediatedPPAR-� activation.

TTA Treatment Markedly Enhances PPAR-�Target Genes in the Liver of Mice but Notin HUVECsTo further examine the role of PPAR-� in the TTA-mediatedeffects on HUVECs and liver, we examined the effect of TTAon some established PPAR-�–target genes. In the mousemodel, TTA enhanced the expression of the liver fattyacid–binding protein (L-FABP) and particularly of CD36 andfatty acyl-coenzyme A oxidase (FAO) in wild-type, but not inPPAR-��/�, mice, underscoring that TTA acts as a PPAR-�agonist in the liver in this mouse model (Figure 4). Incontrast, no such enhancing effects were seen in TNF-�–stimulated HUVECs. In fact, whereas no effects were seen onFAO, TTA tended to decrease CD36 and heart FABP(L-FABP was undetectable) expression in these cells (Figure5).

TTA Decreases Serum Levels of sVCAM-1 andIL-8 in Human patients With PsoriasisOur findings so far suggest antiinflammatory effects of TTAin human endothelial cells in vitro. To further elucidate thepotential in vivo relevance of this finding in human inflam-

Figure 2. The effect of TTA (10 �mol/L, 72 hours) on geneexpression of PPAR-� (A) and PPAR-� (B) in TNF-�–stimulated(10 ng/mL) HUVECs after 6 hours. Data are given asmean�SEM of 3 experiments. mRNA levels were quantified byreal-time RT-PCR and are presented relative to the geneexpression of the house-keeping gene �-actin. *P�0.05 vsunstimulated (Unstim) and TNF-�–stimulated cells.

Figure 3. Wild-type (Wt) and PPAR-��/� mice were treated with 1.7% TTA or standard diet alone for 5 days, and the gene expressionsof VCAM-1 (A), MCP-1 (B), and IL-8 (C) were measured in the liver. Data are given as mean�SEM of 5 experiments. mRNA levels werequantified by real-time RT-PCR and are presented relative to the gene expression of the house-keeping gene GAPDH. *P�0.05 vs dietalone (Control).

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matory disorders, we examined serum levels of sVCAM-1and IL-8 in 43 patients with psoriasis, randomized to TTA orplacebo treatment for 21 days in a double-blind fashion (seeMethods). As shown in Figure 5, sVCAM-1 levels weresignificantly lower in TTA-treated than in placebo-treatedpatients with a similar pattern in male and females (�25%reduction). However, whereas the suppressive effect of TTAon IL-8 levels was even more pronounced than on sVCAM-1in females (�65% reduction), the effect in males was notsignificant (Figure 5). PBMCs were available in 16 of thepatients, and, notably, this antiinflammatory effect of TTAwas also confirmed at the cellular level in PBMCs, showingdownregulated gene expression of MCP-1 and, in particular,TNF-� but not of IL-8, during TTA (P�0.05), but not duringplacebo, therapy. However, the TTA-mediated suppression ofTNF-� and MCP-1 expression was only seen in the female

patients, further supporting a gender-dependent mechanismfor the antiinflammatory effects of TTA in psoriasis. Incontrast to the effect on inflammatory genes, TTA had noeffect on mRNA levels of PPARs in PBMC. Moreover, therewere no differences between sexes in basal levels of lipidsand glucose, in the use of statins, or in PPAR expression inPBMCs (data not shown).

DiscussionThe present study reports that TTA significantly attenuatesTNF-�–induced endothelial cell activation as assessed bydownregulation of IL-8 and VCAM-1 both at mRNA andprotein levels. These effects on adhesion molecules andchemokines were accompanied by a decreased adhesion ofmonocytes to TNF-�–activated endothelium. Moreover, noTTA-mediated attenuation of VCAM-1, IL-8, or MCP-1expression was seen in PPAR-��/� mice, further suggestingthe involvement of this PPAR in the antiinflammatory effectsof TTA. Finally, the relevance of our findings to humandiseases was suggested by a TTA-mediated downregulationof inflammatory mediators in psoriasis patients.

Endothelial dysfunction, manifested as enhanced endothe-lial cell activation, plays an important pathogenic role inseveral vascular and inflammatory disorders.1–3 Activation ofthe endothelium is characterized by increased expression ofadhesion molecules and release of chemokines.3,4,36 Althoughseveral stimuli may be operating, such as microbial antigens,modified autoantigens as well as enhanced oxidative andshear stress, inflammatory cytokines such as TNF-� seem tobe important common mediators in endothelial cell activa-tion.3,4 We have previously shown that TTA increases theTNF-�–mediated release of the antiinflammatory cytokineIL-10 in PBMCs from healthy controls.23 Herein, we showthat TTA treatment significantly attenuates the TNF-�–mediated expression of VCAM-1 and IL-8 in HUVECs,further supporting antiinflammatory effects of this fatty acid.Whether this is attributable to effects of TTA itself or itsoxidative metabolites is at present not entirely clear. How-ever, we currently believe that TTA is the active compound,as oxidation of the saturated acyl-chain of TTA is unlikely tooccur, and no metabolic effects were seen after feeding ratswith tetradecylsulfinyl acetic acid or tetradecylsulfonyl aceticacid.37

Psoriasis is an inflammatory skin disease involving notonly keratinocytes but also endothelial cells and infiltratingleukocytes.38–41 Moreover, upregulation of adhesion mole-cules such as VCAM-1 and enhanced expression of chemo-kines such as IL-8 seems to play an important pathogenic rolein leukocyte trafficking and inflammation in psoriatic der-mis.42–44 Hence, our finding in the present study showing thatTTA significantly downregulated serum levels of sVCAM-1and IL-8 and gene expression of TNF-� in PBMCs inpsoriasis patients underscores the relevance of our findings inHUVECs to human disorders characterized by endothelialcell activation. Kuenzli and Saurat45 have previously sug-gested no effect of TTA in psoriasis, but this study did notconsidered a gender-specific response to TTA. In fact, in thepresent study we found that the downregulatory effect ofTTA on IL-8 and TNF-� was seen only in female patients,

Figure 4. The effect of TTA on PPAR-� target genes. The leftpanels show the effect of pretreatment with TTA (10 �mol/L, 72hours) on the gene expression of CD36 (A), FAO (C), and heartFABP (H-FABP) (E) in TNF-�–stimulated (10 ng/mL) HUVECsafter culturing for 6 hours. The right panels show the effect of1.7% TTA or standard diet alone in 5 days on the gene expres-sion of CD36 (B), FAO (D), and L-FABP (F) in the liver of wild-type (Wt) and PPAR-��/� mice. Data are given as mean�SEMof 3 (HUVEC) or 5 (liver) experiments. mRNA levels were quanti-fied by real-time RT-PCR and are presented relative to the geneexpression of the house-keeping gene �-actin (left panels) orGAPDH (right panels). *P�0.05 vs diet alone (Control). Unstimindicates unstimulated.

Figure 5. Serum levels of IL-8 (A) and sVCAM-1 (B) in psoriasispatients randomized to placebo (n�22) or TTA (n�21, 1 g/d)treatment for 21 days. Data are given as mean�SEM and pre-sented separately for male and females. *P�0.05 vs placebo.

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and a number of factors could contribute to this gender-dependent effect of TTA. Several in vitro and in vivo studiessuggest estrogen-mediated effects on inflammation contrib-uting to sex differences in some inflammatory disorders.46

Even more importantly, with relevance to the present study,there are several reports of cross-talk between estrogenreceptor– and PPAR-signaling pathways.47,48 Moreover, afew studies have shown gender-dependent responses toPPAR agonists.49,50 Nevertheless, relatively few patients werestudied, the follow-up time was rather short, and our resultsfrom the psoriasis study should be interpreted with caution.

In the present study, we found that whereas TTA markedlyenhanced PPAR-�–target genes in the liver of wild-type, butnot of PPAR-��/�, mice, no such effect on PPAR-�–targetgenes was seen in HUVECs. The reasons for these differentresponses in the 2 model systems are at present unclear, butseveral reports suggest that these genes could be regulateddifferently in different organ systems and cell types. Thus, inmice, the expression of CD36 is regulated by PPAR-� in theliver and by PPAR-� in the adipose tissues.51 Moreover, wehave previously reported that TTA can activate all PPARsubtypes,18,20,21 and, in particular, the ability of TTA toactivate PPAR-� may be of relevance for its antiinflammatoryeffects in endothelial cells.52 However, PPAR-independentmechanisms may also be operating. Indeed, we have shownantioxidant effects of TTA that, at least partly, may be PPARindependent, and such effects could clearly be of relevance inthe modulation of TNF-�–activated HUVECs at least partlythrough inhibition of nuclear factor �B activation.53,54 Nev-ertheless, our findings underscore that the antiinflammatoryeffects of TTA seem to involve both PPAR-�–dependent and–independent pathways, at least in some degree differingbetween the different model systems.

In the present study, we used different model systems toexplore the potential immunomodulatory effects of TTA.Although the results cannot necessarily be extrapolated from1 tissue to another, we believe that such an approach will alsohave some advantages. In fact, 1 of our main messages is thatthe effect of TTA, as well as other PPAR agonists, may differamong different tissues, underscoring the complexity whenusing such a therapeutic approach in human disorders. Nev-ertheless, our findings suggest an antiinflammatory potentialof TTA that should be further investigated in disorderscharacterized by persistent inflammation and endothelial cellactivation. In addition to therapeutic trials, such studies willalso have to further clarify the mechanisms of action of TTAin inflammation.

AcknowledgmentsThis work was supported by grants from the Norwegian Associationof Heart and Lung Patients, the University of Bergen, and the IngeMarie Larsine and Gabriel Tidemand Gabrielsens Legacy. Weappreciate the use of equipment from the FFS-MedicalResearch Centre.

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Dyrøy et al Antiinflammatory Effects of TTA 1369

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Methods Preparation of fatty acids

TTA was prepared as previously described 1 by reacting tetradecyl-bromide with thia-acetic

acid and KOH using MeOH as solvent. Through a nucleophilic attack on the α-carbon of

tetradecyl-bromide, TTA and KBr are formed. The product was purified through

crystallization and filtration. Penn Pharmaceuticals (Gwent, UK) manufactured the capsules

used in the psoriasis study (see below). The final product was >99.7% pure, the intra-batch

variation was negligible, and our own gcms-analysis has shown that it is stable for at least 1-2

years at room-temperature. For the cell experiments, TTA was dissolved in dimethyl

sulfoxide to stock solutions of 100 mM. These stock solutions were heated to 37°C and added

to culture medium at final concentrations of 10-100 µM.

Isolation of cells

PBMC and neutrophils were obtained from heparinised blood by Isopaque-Ficoll (PBMC:

Lymphoprep; Nycomed, Oslo, Norway; neutrophils: Polymorphrep; Axis Shieldh, Oslo,

Norway) gradient centrifugation. Further positive selection of CD14+ monocytes were

performed by CD14-labeled magnetic beads (MACS, Milteny Biotec, Bergisch Gladbach,

Germany) as previously described2. After isolation, cell pellets of PBMC were immediately

stored in liquid nitrogen, and the monocytes and neutrophils were used for further

experiments (see below).

Cell experiments

Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from PromoCell (C-

12250; Heidelberg, Germany) and grown in Endothelial Cell Growth Medium (C-22010;

PromoCell). HUVEC, which was used between passages 1 and 4, were maintained in medium

in a humidified chamber containing 5% CO2 at 37°C. Cells were cultured in 25 cm2 or 75 cm2

culture flasks (Sarsted, Nümbrecht, Germany) near confluence and the medium was

exchanged every 48 hours. When studying endothelial cell activation and leukocyte adhesion,

HUVEC of confluent cultures were trypsinized (0.05% wt/vol trypsin and 5 mmol/L EDTA

containing Ca2+ free solution) and seeded at a density of 20,000 cells/well. At day 0, different

concentrations of TTA and after 72 hours of TTA exposure, different concentrations of

human recombinant tumor necrosis factor (TNF)α (Sigma; dissolved in PBS) and PBS were

added to the culture medium. Six and 20 hours thereafter, cell pellets and cell-free

supernatants were harvested and stored at -80ºC. To examine adhesion of monocytes and

neutrophils to HUVEC confluent HUVEC monolayers were grown in 96-well tissue culture

plates (Becton Dickinson Labware, San José, CA). After 72 hours of TTA exposure, half of

the wells were stimulated with TNFα (10 ng/mL) for 24 hours to obtain activated endothelial

cells. After stimulation and washing, freshly isolated monocytes and granulocytes from

healthy human controls were added (1.5 x 105/well) and were allowed to attach for 30 minutes

at 37 °C. Thereafter, non-adherent cells were removed by gently aspiration, and the wells

were washed twice with warm PBS. Adherent cells were fixed in 4% paraformaldehyde for 30

minutes and counted in four separate high-power fields in each well by phase contrast

microscopy (Nikon Phase Contrast-2, Tokyo, Japan). Cell proliferation was assessed by

[3H]Thymidine incorporation as determined by liquid scintillation counting after 4 hours of

incubation with 1.0 µCi/well. The endotoxin levels of all stimulants and culture media were <10

pg/mL (Limulus Amebocyte Assay; BioWhittaker, Walkersville, MD).

Quantitative Real-time RT-PCR

Total RNA was isolated from HUVEC, mouse liver and PBMC using RNeasy Minikit

(Qiagen, Hilden, Germany), and reversed-transcribed using a reverse transcriptase kit

(Applied Biosystems, Foster City, CA). Sequence-specific primers and TaqMan probes were

designed using the Primer Express software version 1.5 (Applied Biosystems; PCR primer

sequences are available on request). Quantification of mRNA was performed using the ABI

Prism7000 (Applied Biosystems) 3. The housekeeping genes β-actin, GAPDH and 18S RNA

were included as endogenous normalization controls to adjust for unequal amounts of RNA.

Similar patterns were obtained whether normalized to β-actin, GAPDH or 18S RNA.

Enzyme immunoassays (EIAs)

IL-8, MCP-1, soluble (s) VCAM-1 and TNFα protein levels were measured by EIAs (R&D

Systems, Minneapolis, MN).

Human psoriasis patients

A total of 43 patients [17 females and 26 males, all Caucasians, mean age 41.4 years (range

22-66 years)] with psoriasis were randomized to placebo or TTA (1g/day) for 21 days in a

double-blind fashion. There were similar key demographic and clinical signs of psoriasis in

the two treatment groups and the majority of the patients were assessed to have psoriasis of

moderate severity (data not shown). The local Ethic Committee and the Norwegian Medicines

Agency approved the study. Signed informed consent was obtained from each patient.

Animal studies

The animal study has been previously described,4. Briefly, wild-type and PPARα-/- mice (20-

25 g), were pure-bred on a sv129 background 5. The mice were given standard pellets [21.8%

casein, 10% soy oil, 17% vitamin/mineral mixture (5.83% vitamin mixture, AIN-93VX, Dyets

Inc.; 17.4% mineral mixture, AIN-93G, Dyets Inc.; 11.6% cellulose; 63,9% sucrose; 1.2%

cholintartrate) and dextrin (49.5–51.2%)] alone or supplemented with 1.7% TTA. At

termination the animals were anesthetized with a subcutaneous injection of Hypnorm

Dormicum (fentanyl/fluanisone midazol-am, 0.2 ml/100 g body weight). Livers were either

immediately removed, placed in ice-cold homogenizing medium and weighed, or freeze-

clamped in situ and stored in liquid nitrogen. The Norwegian State Board of Biological

Experiments with Living Animals approved the protocol.

Statistics

Each HUVEC experiment was performed in duplicates or triplicates (growth experiments

with 8 parallels), and each experiment was repeated at least three times. Differences between

groups were tested by Student t test. For comparison of human data, the Mann-Whitney U test

was used. Probability values (2-sided) were considered significant at value of <0.05.

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4. Grav HJ, Tronstad KJ, Gudbrandsen OA, Berge K, Fladmark KE, Martinsen TC, Waldum H, Wergedahl H, Berge RK. Changed energy state and increased mitochondrial beta-oxidation rate in liver of rats associated with lowered proton electrochemical potential and stimulated uncoupling protein 2 (UCP-2) expression: evidence for peroxisome proliferator-activated receptor-α independent induction of UCP-2 expression. J Biol Chem. 2003;278:30525-30533.

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Figure Legends Figure I. Panel A shows the effects of various concentrations of TNFα on the mRNA levels

of E-selectin, IL-8 and VCAM-1 in HUVEC after culturing for 6 hours. Panel B shows the

time-dependent effects of TNFα (10 ng/ml) on the gene expression of these mediators in

HUVEC. Data are given as mean±SEM of 3 experiments. mRNA levels were quantified by

real-time RT-PCR and are presented relative to the gene expression of the house-keeping gene

β-actin.

and Rolf K. BergeEndre Dyrøy, Arne Yndestad, Thor Ueland, Bente Halvorsen, Jan Kristian Damås, Pål Aukrust

Independent Pathways−Dependent and −αActivated Receptor −Proliferator Antiinflammatory Effects of Tetradecylthioacetic Acid Involve Both Peroxisome

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