Antiinflammatory Effects of Tetradecylthioacetic Acid Involve Both Peroxisome Proliferator–Activated Receptor –Dependent and –Independent Pa
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动脉硬化血栓血管生物学 2005年第7期
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 and Infectious Diseases (P.A.), Medical Department, Rikshospitalet, University of Oslo, Oslo, Norway.
Correspondence to Endre Dyr?y, Section of Medical Biochemistry, Institute of Medicine University of Bergen, Haukeland University Hospital N-5021, Bergen, Norway. E-mail endre.dyroy@med.uib.no
Abstract
Objective— Tetradecylthioacetic acid (TTA) is a hypolipidemic antioxidant with immunomodulating properties involving activation of peroxisome proliferator–activated receptors (PPARs). Human endothelial cells express PPARs. We hypothesized 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 studies in human endothelial cells (HUVECs) and in vivo studies in humans and PPAR-–/– mice. Our main findings were as follows: (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 chemokine expression was seen in the liver of PPAR-–/– mice. (3) Whereas TTA markedly enhanced PPAR-–target genes in the liver of wild-type, but not of PPAR-–/–, mice, no such effect on PPAR-–target genes was seen in HUVECs. (4) The relevance of our findings to human disease was suggested by a TTA-mediated downregulation of serum levels of soluble VCAM-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.
We show that tetradecylthioacetic acid attenuates tumor necrosis factor-–mediated endothelial cell activation, supporting antiinflammatory effects of this fatty acid. The relevance of our findings to human diseases was suggested by a tetradecylthioacetic acid–mediated downregulation of inflammatory mediators in psoriasis patients.
Key Words: endothelial dysfunction ? inflammation ? cytokine ? peroxisome proliferator–activated receptors
Introduction
Enhanced activation of the endothelium seems to play a pathogenic role in several inflammatory disorders such as atherosclerosis, diabetes, and various autoimmune disorders.1–3 Endothelial cell activation refers to changes in the endothelium as a result of, for example, cytokine stimulation during inflammatory and infectious conditions.4,5 Typically, these changes include expression of adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1),6 and chemokines, such as monocyte chemoattractant protein 1 (MCP-1) and interleukin 8 (IL-8).7–9 The end result is recruitment of leukocytes with transmigration of cells into the arterial wall promoting local inflammation and further recruitment of activated leukocytes, representing a pathogenic loop in several inflammatory disorders.10 Accordingly, endothelial cell activation represents an interesting pharmaceutical target for prevention and modulation of various inflammatory and cardiovascular diseases.11,12
3-Thia fatty acids such as tetradecylthioacetic acid (TTA), are modified fatty acids that promote hepatic proliferation of mitochondria and peroxisomes and decrease serum triacylglycerol, cholesterol, and free fatty acid levels in animal models.13–15 The chemical properties of TTA are similar to normal fatty acids of similar length, but the metabolism and metabolic effects of TTA differ markedly from these other fatty acids.16,17 Whereas normal ?-oxidation of TTA does not occur, it is catabolized through -oxidation and sulfur oxidation, and short dicarboxylic metabolites can be found in the urine of animals and patients receiving TTA. TTA can also be desaturated, and a small proportion of its 9-desaturated metabolite is found in plasma and tissues of patients and animals treated with TTA.
The hypolipidemic response after TTA treatment seems to involve activation of peroxisome proliferator–activated receptors (PPARs) in the liver.18,19 However, it has been shown that TTA is a ligand for all PPAR subtypes,20,21 indicating that TTA could also act through PPARs outside the liver. Moreover, TTA has been shown to have antioxidant effects in vitro,22 and we demonstrated recently that TTA also has immunomodulatory properties in human peripheral blood mononuclear cells (PBMCs)23 and in HIV-infected patients.24 However, it is not known whether TTA can modulate endothelial cell activation.
Human endothelial cells express both PPAR- and PPAR-,25,26 and activation of PPAR-, but not PPAR-,25 inhibits cytokine-induced VCAM-1 expression in human endothelial cells. We hypothesized that TTA could modulate endothelial cell activation and inflammation at least partly involving PPAR-related mechanisms. In the present study, this hypothesis was explored by different experimental approaches involving in vitro studies in human endothelial cells, in vivo studies in psoriasis patients, and PPAR-–/– mice.
Methods
An online Methods section is available in the data supplement at http://atvb.ahajournals.org.
Preparation of Fatty Acids
TTA was prepared as previously described.27 Penn Pharmaceuticals (Gwent, UK) manufactured the capsules used in the psoriasis study.
Cell Experiments
PBMCs and neutrophils were obtained from heparinized blood by Isopaque-Ficoll (PBMCs: Lymphoprep, Nycomed, Oslo, Norway; neutrophils: Polymorphrep, Axis Shieldh, Oslo) gradient centrifugation. Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (C-12250; Heidelberg, Germany). Cell proliferation was assessed by [3H]thymidine incorporation.
Quantitative Real-Time Reverse Transcription–Polymerase Chain Reaction
Total RNA was isolated from HUVECs, mouse liver, and PBMCs using RNeasy Minikit (Qiagen, Hilden, Germany) and reversed-transcribed using a reverse transcriptase kit (Applied Biosystems, Foster City, Calif). Quantification of mRNA was performed using the ABI Prism7000 (Applied Biosystems).29
Enzyme Immunoassays
IL-8, MCP-1, soluble VCAM-1 (sVCAM-1), and tumor necrosis factor (TNF-) protein levels were measured by enzyme immunoassay (R&D Systems, Minneapolis).
Human Psoriasis Patients
A 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 to placebo or TTA (1 g/d) 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 ethics 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.30 Briefly, wild-type and PPAR-–/– mice (20 to 25 g) were pure-bred on a SV129 background.31 The Norwegian State Board of Biological Experiments with Living Animals approved the protocol.
Statistics
Each HUVEC experiment was performed in duplicate or triplicate (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 P<0.05.
Results
TNF- Activates Endothelial Cells in a Dose- and Time-Dependent Manner
TNF- markedly enhanced the expression of VCAM-1, IL-8, and endothelial (E-selectin) in HUVECs in a dose-dependent manner, reaching a plateau at 1 ng/mL for IL-8 and 5 ng/mL for VCAM-1 and E-Selectin (Figure IA, available online at http://atvb.ahajournals.org). Time-course experiments showed a marked TNF-–induced expression of these genes already after 3 hours (Figure IB). However, whereas IL-8 and VCAM-1 mRNA levels continued to rise during the observation period, reaching maximum after 20 hours, E-selectin expression declined after 3 hours of TNF- exposure (Figure IB).
TTA Reduces TNF-–Induced Endothelial Activation
Pretreatment with TTA attenuated the TNF-–induced mRNA expression of VCAM-1 and IL-8 but not of E-selectin, with suppressive effects of TTA at concentrations 10 μmol/L (Figure 1A). A similar pattern was also seen at the protein level in HUVEC supernatants, showing a suppressive effect of TTA (10 μmol/L) on the TNF-–induced release of sVCAM-1 and, in particular, of IL-8 (Figure 1B and 1C). In fact, TTA nearly abolished completely the TNF-–induced release of IL-8. TTA has previously been shown to inhibit cell growth and induce apoptosis in cancer cells21,32,33 and smooth muscle cells.34 However, TTA did not reduce growth of endothelial cells at dosages up to 100 μmol/L, as assessed by [3H]thymidine incorporation and had no effect on total protein synthesis or mRNA concentrations (data not shown).
Figure 1. A, Effect of different concentrations of TTA (pretreatment for 72 hours) on the gene expression of E-selectin, IL-8, and VCAM-1 in TNF-–stimulated HUVECs after culturing for 6 hours. Data are given as mean±SEM of 3 experiments. mRNA levels were quantified by real-time reverse transcription–polymerase chain reaction (RT-PCR) and are presented relative to the gene expression of the house-keeping gene ?-actin. B and C, Effect of TTA (10 μmol/L) on the release of IL-8 and sVCAM-1, respectively, in TNF-–stimulated (10 ng/mL) HUVEC supernatants after culturing for 20 hours as assessed by enzyme immunoassay. Data are given as mean±SEM of 3 experiments. D, Effect of TTA (10 μmol/L) on the adhesion of monocytes and neutrophils to TNF-–activated (10 ng/mL) HUVECs. Data are given as mean±SEM of 6 experiments. *P<0.05 vs TNF-–stimulated cells for all panels. Unstim indicates unstimulated.
Effect of TTA on TNF-–Induced Adhesion of Monocytes and Neutrophils to HUVECs
It has previously been reported that certain PPAR agonists may help limit chronic inflammation mediated by VCAM-1 and monocytes, without affecting acute inflammation mediated by E-selectin and neutrophil binding.35 Based on the ability of TTA to downregulate VCAM-1, but not E-selectin, in HUVECs, we examined whether TTA modulated differently the adhesion of neutrophils and monocytes to TNF-–activated HUVECs. As shown in Figure 1D, whereas TTA significantly attenuated monocyte adhesion to TNF-–activated HUVECs, no such effect was seen on neutrophil adhesion.
TTA Increases Both PPAR- and PPAR- Gene Expression in Endothelial Cells
To 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 PPARs in these cells. Notably, we found that TTA treatment enhanced mRNA level of both PPAR- (2.5 fold) and PPAR- (1.7-fold) in TNF-–treated HUVECs (Figure 2). The PPAR- gene expression was too weak to yield reliable quantitative results.
Figure 2. The effect of TTA (10 μmol/L, 72 hours) on gene expression of PPAR- (A) and PPAR- (B) in TNF-–stimulated (10 ng/mL) HUVECs after 6 hours. 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 ?-actin. *P<0.05 vs unstimulated (Unstim) and TNF-–stimulated cells.
TTA Treatment Does Not Attenuate VCAM-1, IL-8, or MCP-1 Expression in PPAR-–/– Mice
To further elucidate the role of PPARs in the antiinflammatory effects of TTA, we examined the ability of TTA to modify the expression of VCAM-1, IL-8, and MCP-1 in the liver from wild-type and PPAR-–/– mice. As shown in Figure 3, TTA suppressed both VCAM-1 and, in particular, IL-8 and MCP-1 expression in the liver of wild-type mice. In contrast, the opposite effect was seen in PPAR-–/– mice with an enhancing effect of TTA on these mediators (Figure 3). The reason for this latter finding is not clear but may reflect that some PPAR-–independent enhancing effects of TTA on these mediators are counter-acted by TTA-mediated PPAR- activation.
Figure 3. Wild-type (Wt) and PPAR-–/– mice were treated with 1.7% TTA or standard diet alone for 5 days, and the gene expressions of 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 were quantified by real-time RT-PCR and are presented relative to the gene expression of the house-keeping gene GAPDH. *P<0.05 vs diet alone (Control).
TTA Treatment Markedly Enhances PPAR- Target Genes in the Liver of Mice but Not in HUVECs
To further examine the role of PPAR- in the TTA-mediated effects on HUVECs and liver, we examined the effect of TTA on some established PPAR-–target genes. In the mouse model, TTA enhanced the expression of the liver fatty acid–binding protein (L-FABP) and particularly of CD36 and fatty acyl-coenzyme A oxidase (FAO) in wild-type, but not in PPAR-–/–, mice, underscoring that TTA acts as a PPAR- agonist in the liver in this mouse model (Figure 4). In contrast, no such enhancing effects were seen in TNF-–stimulated HUVECs. In fact, whereas no effects were seen on FAO, TTA tended to decrease CD36 and heart FABP (L-FABP was undetectable) expression in these cells (Figure 5).
Figure 4. The effect of TTA on PPAR- target genes. The left panels show the effect of pretreatment with TTA (10 μmol/L, 72 hours) on the gene expression of CD36 (A), FAO (C), and heart FABP (H-FABP) (E) in TNF-–stimulated (10 ng/mL) HUVECs after culturing for 6 hours. The right panels show the effect of 1.7% TTA or standard diet alone in 5 days on the gene expression of CD36 (B), FAO (D), and L-FABP (F) in the liver of wild-type (Wt) and PPAR-–/– mice. Data are given as mean±SEM of 3 (HUVEC) or 5 (liver) experiments. mRNA levels were quantified by real-time RT-PCR and are presented relative to the gene expression of the house-keeping gene ?-actin (left panels) or GAPDH (right panels). *P<0.05 vs diet alone (Control). Unstim indicates unstimulated.
Figure 5. Serum levels of IL-8 (A) and sVCAM-1 (B) in psoriasis patients randomized to placebo (n=22) or TTA (n=21, 1 g/d) treatment for 21 days. Data are given as mean±SEM and presented separately for male and females. *P<0.05 vs placebo.
TTA Decreases Serum Levels of sVCAM-1 and IL-8 in Human patients With Psoriasis
Our findings so far suggest antiinflammatory effects of TTA in human endothelial cells in vitro. To further elucidate the potential in vivo relevance of this finding in human inflammatory disorders, we examined serum levels of sVCAM-1 and IL-8 in 43 patients with psoriasis, randomized to TTA or placebo treatment for 21 days in a double-blind fashion (see Methods). As shown in Figure 5, sVCAM-1 levels were significantly lower in TTA-treated than in placebo-treated patients with a similar pattern in male and females (25% reduction). However, whereas the suppressive effect of TTA on IL-8 levels was even more pronounced than on sVCAM-1 in females (65% reduction), the effect in males was not significant (Figure 5). PBMCs were available in 16 of the patients, and, notably, this antiinflammatory effect of TTA was also confirmed at the cellular level in PBMCs, showing downregulated gene expression of MCP-1 and, in particular, TNF- but not of IL-8, during TTA (P<0.05), but not during placebo, therapy. However, the TTA-mediated suppression of TNF- and MCP-1 expression was only seen in the female patients, further supporting a gender-dependent mechanism for the antiinflammatory effects of TTA in psoriasis. In contrast to the effect on inflammatory genes, TTA had no effect on mRNA levels of PPARs in PBMC. Moreover, there were no differences between sexes in basal levels of lipids and glucose, in the use of statins, or in PPAR expression in PBMCs (data not shown).
Discussion
The present study reports that TTA significantly attenuates TNF-–induced endothelial cell activation as assessed by downregulation of IL-8 and VCAM-1 both at mRNA and protein levels. These effects on adhesion molecules and chemokines were accompanied by a decreased adhesion of monocytes to TNF-–activated endothelium. Moreover, no TTA-mediated attenuation of VCAM-1, IL-8, or MCP-1 expression was seen in PPAR-–/– mice, further suggesting the involvement of this PPAR in the antiinflammatory effects of TTA. Finally, the relevance of our findings to human diseases was suggested by a TTA-mediated downregulation of inflammatory mediators in psoriasis patients.
Endothelial dysfunction, manifested as enhanced endothelial cell activation, plays an important pathogenic role in several vascular and inflammatory disorders.1–3 Activation of the endothelium is characterized by increased expression of adhesion molecules and release of chemokines.3,4,36 Although several stimuli may be operating, such as microbial antigens, modified autoantigens as well as enhanced oxidative and shear stress, inflammatory cytokines such as TNF- seem to be important common mediators in endothelial cell activation.3,4 We have previously shown that TTA increases the TNF-–mediated release of the antiinflammatory cytokine IL-10 in PBMCs from healthy controls.23 Herein, we show that 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 its oxidative metabolites is at present not entirely clear. However, we currently believe that TTA is the active compound, as oxidation of the saturated acyl-chain of TTA is unlikely to occur, and no metabolic effects were seen after feeding rats with tetradecylsulfinyl acetic acid or tetradecylsulfonyl acetic acid.37
Psoriasis is an inflammatory skin disease involving not only keratinocytes but also endothelial cells and infiltrating leukocytes.38–41 Moreover, upregulation of adhesion molecules such as VCAM-1 and enhanced expression of chemokines such as IL-8 seems to play an important pathogenic role in leukocyte trafficking and inflammation in psoriatic dermis.42–44 Hence, our finding in the present study showing that TTA significantly downregulated serum levels of sVCAM-1 and IL-8 and gene expression of TNF- in PBMCs in psoriasis patients underscores the relevance of our findings in HUVECs to human disorders characterized by endothelial cell activation. Kuenzli and Saurat45 have previously suggested no effect of TTA in psoriasis, but this study did not considered a gender-specific response to TTA. In fact, in the present study we found that the downregulatory effect of TTA on IL-8 and TNF- was seen only in female patients, and a number of factors could contribute to this gender-dependent effect of TTA. Several in vitro and in vivo studies suggest estrogen-mediated effects on inflammation contributing 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 estrogen receptor– and PPAR-signaling pathways.47,48 Moreover, a few studies have shown gender-dependent responses to PPAR agonists.49,50 Nevertheless, relatively few patients were studied, the follow-up time was rather short, and our results from the psoriasis study should be interpreted with caution.
In the present study, we found that whereas TTA markedly enhanced PPAR-–target genes in the liver of wild-type, but not of PPAR-–/–, mice, no such effect on PPAR-–target genes was seen in HUVECs. The reasons for these different responses in the 2 model systems are at present unclear, but several reports suggest that these genes could be regulated differently in different organ systems and cell types. Thus, in mice, the expression of CD36 is regulated by PPAR- in the liver and by PPAR- in the adipose tissues.51 Moreover, we have previously reported that TTA can activate all PPAR subtypes,18,20,21 and, in particular, the ability of TTA to activate PPAR- may be of relevance for its antiinflammatory effects in endothelial cells.52 However, PPAR-independent mechanisms may also be operating. Indeed, we have shown antioxidant effects of TTA that, at least partly, may be PPAR independent, and such effects could clearly be of relevance in the modulation of TNF-–activated HUVECs at least partly through inhibition of nuclear factor B activation.53,54 Nevertheless, our findings underscore that the antiinflammatory effects of TTA seem to involve both PPAR-–dependent and –independent pathways, at least in some degree differing between the different model systems.
In the present study, we used different model systems to explore the potential immunomodulatory effects of TTA. Although the results cannot necessarily be extrapolated from 1 tissue to another, we believe that such an approach will also have some advantages. In fact, 1 of our main messages is that the effect of TTA, as well as other PPAR agonists, may differ among different tissues, underscoring the complexity when using such a therapeutic approach in human disorders. Nevertheless, our findings suggest an antiinflammatory potential of TTA that should be further investigated in disorders characterized by persistent inflammation and endothelial cell activation. In addition to therapeutic trials, such studies will also have to further clarify the mechanisms of action of TTA in inflammation.
Acknowledgments
This work was supported by grants from the Norwegian Association of Heart and Lung Patients, the University of Bergen, and the Inge Marie Larsine and Gabriel Tidemand Gabrielsens Legacy. We appreciate the use of equipment from the FFS-Medical Research Centre.
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Delerive P, Gervois P, Fruchart JC, Staels B. Induction of IB expression as a mechanism contributing to the antiinflammatory activities of peroxisome proliferator-activated receptor- activators. J Biol Chem. 2000; 275: 36703–36707.(Endre Dyr?y; Arne Yndesta)
Correspondence to Endre Dyr?y, Section of Medical Biochemistry, Institute of Medicine University of Bergen, Haukeland University Hospital N-5021, Bergen, Norway. E-mail endre.dyroy@med.uib.no
Abstract
Objective— Tetradecylthioacetic acid (TTA) is a hypolipidemic antioxidant with immunomodulating properties involving activation of peroxisome proliferator–activated receptors (PPARs). Human endothelial cells express PPARs. We hypothesized 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 studies in human endothelial cells (HUVECs) and in vivo studies in humans and PPAR-–/– mice. Our main findings were as follows: (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 chemokine expression was seen in the liver of PPAR-–/– mice. (3) Whereas TTA markedly enhanced PPAR-–target genes in the liver of wild-type, but not of PPAR-–/–, mice, no such effect on PPAR-–target genes was seen in HUVECs. (4) The relevance of our findings to human disease was suggested by a TTA-mediated downregulation of serum levels of soluble VCAM-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.
We show that tetradecylthioacetic acid attenuates tumor necrosis factor-–mediated endothelial cell activation, supporting antiinflammatory effects of this fatty acid. The relevance of our findings to human diseases was suggested by a tetradecylthioacetic acid–mediated downregulation of inflammatory mediators in psoriasis patients.
Key Words: endothelial dysfunction ? inflammation ? cytokine ? peroxisome proliferator–activated receptors
Introduction
Enhanced activation of the endothelium seems to play a pathogenic role in several inflammatory disorders such as atherosclerosis, diabetes, and various autoimmune disorders.1–3 Endothelial cell activation refers to changes in the endothelium as a result of, for example, cytokine stimulation during inflammatory and infectious conditions.4,5 Typically, these changes include expression of adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1),6 and chemokines, such as monocyte chemoattractant protein 1 (MCP-1) and interleukin 8 (IL-8).7–9 The end result is recruitment of leukocytes with transmigration of cells into the arterial wall promoting local inflammation and further recruitment of activated leukocytes, representing a pathogenic loop in several inflammatory disorders.10 Accordingly, endothelial cell activation represents an interesting pharmaceutical target for prevention and modulation of various inflammatory and cardiovascular diseases.11,12
3-Thia fatty acids such as tetradecylthioacetic acid (TTA), are modified fatty acids that promote hepatic proliferation of mitochondria and peroxisomes and decrease serum triacylglycerol, cholesterol, and free fatty acid levels in animal models.13–15 The chemical properties of TTA are similar to normal fatty acids of similar length, but the metabolism and metabolic effects of TTA differ markedly from these other fatty acids.16,17 Whereas normal ?-oxidation of TTA does not occur, it is catabolized through -oxidation and sulfur oxidation, and short dicarboxylic metabolites can be found in the urine of animals and patients receiving TTA. TTA can also be desaturated, and a small proportion of its 9-desaturated metabolite is found in plasma and tissues of patients and animals treated with TTA.
The hypolipidemic response after TTA treatment seems to involve activation of peroxisome proliferator–activated receptors (PPARs) in the liver.18,19 However, it has been shown that TTA is a ligand for all PPAR subtypes,20,21 indicating that TTA could also act through PPARs outside the liver. Moreover, TTA has been shown to have antioxidant effects in vitro,22 and we demonstrated recently that TTA also has immunomodulatory properties in human peripheral blood mononuclear cells (PBMCs)23 and in HIV-infected patients.24 However, it is not known whether TTA can modulate endothelial cell activation.
Human endothelial cells express both PPAR- and PPAR-,25,26 and activation of PPAR-, but not PPAR-,25 inhibits cytokine-induced VCAM-1 expression in human endothelial cells. We hypothesized that TTA could modulate endothelial cell activation and inflammation at least partly involving PPAR-related mechanisms. In the present study, this hypothesis was explored by different experimental approaches involving in vitro studies in human endothelial cells, in vivo studies in psoriasis patients, and PPAR-–/– mice.
Methods
An online Methods section is available in the data supplement at http://atvb.ahajournals.org.
Preparation of Fatty Acids
TTA was prepared as previously described.27 Penn Pharmaceuticals (Gwent, UK) manufactured the capsules used in the psoriasis study.
Cell Experiments
PBMCs and neutrophils were obtained from heparinized blood by Isopaque-Ficoll (PBMCs: Lymphoprep, Nycomed, Oslo, Norway; neutrophils: Polymorphrep, Axis Shieldh, Oslo) gradient centrifugation. Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (C-12250; Heidelberg, Germany). Cell proliferation was assessed by [3H]thymidine incorporation.
Quantitative Real-Time Reverse Transcription–Polymerase Chain Reaction
Total RNA was isolated from HUVECs, mouse liver, and PBMCs using RNeasy Minikit (Qiagen, Hilden, Germany) and reversed-transcribed using a reverse transcriptase kit (Applied Biosystems, Foster City, Calif). Quantification of mRNA was performed using the ABI Prism7000 (Applied Biosystems).29
Enzyme Immunoassays
IL-8, MCP-1, soluble VCAM-1 (sVCAM-1), and tumor necrosis factor (TNF-) protein levels were measured by enzyme immunoassay (R&D Systems, Minneapolis).
Human Psoriasis Patients
A 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 to placebo or TTA (1 g/d) 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 ethics 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.30 Briefly, wild-type and PPAR-–/– mice (20 to 25 g) were pure-bred on a SV129 background.31 The Norwegian State Board of Biological Experiments with Living Animals approved the protocol.
Statistics
Each HUVEC experiment was performed in duplicate or triplicate (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 P<0.05.
Results
TNF- Activates Endothelial Cells in a Dose- and Time-Dependent Manner
TNF- markedly enhanced the expression of VCAM-1, IL-8, and endothelial (E-selectin) in HUVECs in a dose-dependent manner, reaching a plateau at 1 ng/mL for IL-8 and 5 ng/mL for VCAM-1 and E-Selectin (Figure IA, available online at http://atvb.ahajournals.org). Time-course experiments showed a marked TNF-–induced expression of these genes already after 3 hours (Figure IB). However, whereas IL-8 and VCAM-1 mRNA levels continued to rise during the observation period, reaching maximum after 20 hours, E-selectin expression declined after 3 hours of TNF- exposure (Figure IB).
TTA Reduces TNF-–Induced Endothelial Activation
Pretreatment with TTA attenuated the TNF-–induced mRNA expression of VCAM-1 and IL-8 but not of E-selectin, with suppressive effects of TTA at concentrations 10 μmol/L (Figure 1A). A similar pattern was also seen at the protein level in HUVEC supernatants, showing a suppressive effect of TTA (10 μmol/L) on the TNF-–induced release of sVCAM-1 and, in particular, of IL-8 (Figure 1B and 1C). In fact, TTA nearly abolished completely the TNF-–induced release of IL-8. TTA has previously been shown to inhibit cell growth and induce apoptosis in cancer cells21,32,33 and smooth muscle cells.34 However, TTA did not reduce growth of endothelial cells at dosages up to 100 μmol/L, as assessed by [3H]thymidine incorporation and had no effect on total protein synthesis or mRNA concentrations (data not shown).
Figure 1. A, Effect of different concentrations of TTA (pretreatment for 72 hours) on the gene expression of E-selectin, IL-8, and VCAM-1 in TNF-–stimulated HUVECs after culturing for 6 hours. Data are given as mean±SEM of 3 experiments. mRNA levels were quantified by real-time reverse transcription–polymerase chain reaction (RT-PCR) and are presented relative to the gene expression of the house-keeping gene ?-actin. B and C, Effect of TTA (10 μmol/L) on the release of IL-8 and sVCAM-1, respectively, in TNF-–stimulated (10 ng/mL) HUVEC supernatants after culturing for 20 hours as assessed by enzyme immunoassay. Data are given as mean±SEM of 3 experiments. D, Effect of TTA (10 μmol/L) on the adhesion of monocytes and neutrophils to TNF-–activated (10 ng/mL) HUVECs. Data are given as mean±SEM of 6 experiments. *P<0.05 vs TNF-–stimulated cells for all panels. Unstim indicates unstimulated.
Effect of TTA on TNF-–Induced Adhesion of Monocytes and Neutrophils to HUVECs
It has previously been reported that certain PPAR agonists may help limit chronic inflammation mediated by VCAM-1 and monocytes, without affecting acute inflammation mediated by E-selectin and neutrophil binding.35 Based on the ability of TTA to downregulate VCAM-1, but not E-selectin, in HUVECs, we examined whether TTA modulated differently the adhesion of neutrophils and monocytes to TNF-–activated HUVECs. As shown in Figure 1D, whereas TTA significantly attenuated monocyte adhesion to TNF-–activated HUVECs, no such effect was seen on neutrophil adhesion.
TTA Increases Both PPAR- and PPAR- Gene Expression in Endothelial Cells
To 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 PPARs in these cells. Notably, we found that TTA treatment enhanced mRNA level of both PPAR- (2.5 fold) and PPAR- (1.7-fold) in TNF-–treated HUVECs (Figure 2). The PPAR- gene expression was too weak to yield reliable quantitative results.
Figure 2. The effect of TTA (10 μmol/L, 72 hours) on gene expression of PPAR- (A) and PPAR- (B) in TNF-–stimulated (10 ng/mL) HUVECs after 6 hours. 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 ?-actin. *P<0.05 vs unstimulated (Unstim) and TNF-–stimulated cells.
TTA Treatment Does Not Attenuate VCAM-1, IL-8, or MCP-1 Expression in PPAR-–/– Mice
To further elucidate the role of PPARs in the antiinflammatory effects of TTA, we examined the ability of TTA to modify the expression of VCAM-1, IL-8, and MCP-1 in the liver from wild-type and PPAR-–/– mice. As shown in Figure 3, TTA suppressed both VCAM-1 and, in particular, IL-8 and MCP-1 expression in the liver of wild-type mice. In contrast, the opposite effect was seen in PPAR-–/– mice with an enhancing effect of TTA on these mediators (Figure 3). The reason for this latter finding is not clear but may reflect that some PPAR-–independent enhancing effects of TTA on these mediators are counter-acted by TTA-mediated PPAR- activation.
Figure 3. Wild-type (Wt) and PPAR-–/– mice were treated with 1.7% TTA or standard diet alone for 5 days, and the gene expressions of 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 were quantified by real-time RT-PCR and are presented relative to the gene expression of the house-keeping gene GAPDH. *P<0.05 vs diet alone (Control).
TTA Treatment Markedly Enhances PPAR- Target Genes in the Liver of Mice but Not in HUVECs
To further examine the role of PPAR- in the TTA-mediated effects on HUVECs and liver, we examined the effect of TTA on some established PPAR-–target genes. In the mouse model, TTA enhanced the expression of the liver fatty acid–binding protein (L-FABP) and particularly of CD36 and fatty acyl-coenzyme A oxidase (FAO) in wild-type, but not in PPAR-–/–, mice, underscoring that TTA acts as a PPAR- agonist in the liver in this mouse model (Figure 4). In contrast, no such enhancing effects were seen in TNF-–stimulated HUVECs. In fact, whereas no effects were seen on FAO, TTA tended to decrease CD36 and heart FABP (L-FABP was undetectable) expression in these cells (Figure 5).
Figure 4. The effect of TTA on PPAR- target genes. The left panels show the effect of pretreatment with TTA (10 μmol/L, 72 hours) on the gene expression of CD36 (A), FAO (C), and heart FABP (H-FABP) (E) in TNF-–stimulated (10 ng/mL) HUVECs after culturing for 6 hours. The right panels show the effect of 1.7% TTA or standard diet alone in 5 days on the gene expression of CD36 (B), FAO (D), and L-FABP (F) in the liver of wild-type (Wt) and PPAR-–/– mice. Data are given as mean±SEM of 3 (HUVEC) or 5 (liver) experiments. mRNA levels were quantified by real-time RT-PCR and are presented relative to the gene expression of the house-keeping gene ?-actin (left panels) or GAPDH (right panels). *P<0.05 vs diet alone (Control). Unstim indicates unstimulated.
Figure 5. Serum levels of IL-8 (A) and sVCAM-1 (B) in psoriasis patients randomized to placebo (n=22) or TTA (n=21, 1 g/d) treatment for 21 days. Data are given as mean±SEM and presented separately for male and females. *P<0.05 vs placebo.
TTA Decreases Serum Levels of sVCAM-1 and IL-8 in Human patients With Psoriasis
Our findings so far suggest antiinflammatory effects of TTA in human endothelial cells in vitro. To further elucidate the potential in vivo relevance of this finding in human inflammatory disorders, we examined serum levels of sVCAM-1 and IL-8 in 43 patients with psoriasis, randomized to TTA or placebo treatment for 21 days in a double-blind fashion (see Methods). As shown in Figure 5, sVCAM-1 levels were significantly lower in TTA-treated than in placebo-treated patients with a similar pattern in male and females (25% reduction). However, whereas the suppressive effect of TTA on IL-8 levels was even more pronounced than on sVCAM-1 in females (65% reduction), the effect in males was not significant (Figure 5). PBMCs were available in 16 of the patients, and, notably, this antiinflammatory effect of TTA was also confirmed at the cellular level in PBMCs, showing downregulated gene expression of MCP-1 and, in particular, TNF- but not of IL-8, during TTA (P<0.05), but not during placebo, therapy. However, the TTA-mediated suppression of TNF- and MCP-1 expression was only seen in the female patients, further supporting a gender-dependent mechanism for the antiinflammatory effects of TTA in psoriasis. In contrast to the effect on inflammatory genes, TTA had no effect on mRNA levels of PPARs in PBMC. Moreover, there were no differences between sexes in basal levels of lipids and glucose, in the use of statins, or in PPAR expression in PBMCs (data not shown).
Discussion
The present study reports that TTA significantly attenuates TNF-–induced endothelial cell activation as assessed by downregulation of IL-8 and VCAM-1 both at mRNA and protein levels. These effects on adhesion molecules and chemokines were accompanied by a decreased adhesion of monocytes to TNF-–activated endothelium. Moreover, no TTA-mediated attenuation of VCAM-1, IL-8, or MCP-1 expression was seen in PPAR-–/– mice, further suggesting the involvement of this PPAR in the antiinflammatory effects of TTA. Finally, the relevance of our findings to human diseases was suggested by a TTA-mediated downregulation of inflammatory mediators in psoriasis patients.
Endothelial dysfunction, manifested as enhanced endothelial cell activation, plays an important pathogenic role in several vascular and inflammatory disorders.1–3 Activation of the endothelium is characterized by increased expression of adhesion molecules and release of chemokines.3,4,36 Although several stimuli may be operating, such as microbial antigens, modified autoantigens as well as enhanced oxidative and shear stress, inflammatory cytokines such as TNF- seem to be important common mediators in endothelial cell activation.3,4 We have previously shown that TTA increases the TNF-–mediated release of the antiinflammatory cytokine IL-10 in PBMCs from healthy controls.23 Herein, we show that 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 its oxidative metabolites is at present not entirely clear. However, we currently believe that TTA is the active compound, as oxidation of the saturated acyl-chain of TTA is unlikely to occur, and no metabolic effects were seen after feeding rats with tetradecylsulfinyl acetic acid or tetradecylsulfonyl acetic acid.37
Psoriasis is an inflammatory skin disease involving not only keratinocytes but also endothelial cells and infiltrating leukocytes.38–41 Moreover, upregulation of adhesion molecules such as VCAM-1 and enhanced expression of chemokines such as IL-8 seems to play an important pathogenic role in leukocyte trafficking and inflammation in psoriatic dermis.42–44 Hence, our finding in the present study showing that TTA significantly downregulated serum levels of sVCAM-1 and IL-8 and gene expression of TNF- in PBMCs in psoriasis patients underscores the relevance of our findings in HUVECs to human disorders characterized by endothelial cell activation. Kuenzli and Saurat45 have previously suggested no effect of TTA in psoriasis, but this study did not considered a gender-specific response to TTA. In fact, in the present study we found that the downregulatory effect of TTA on IL-8 and TNF- was seen only in female patients, and a number of factors could contribute to this gender-dependent effect of TTA. Several in vitro and in vivo studies suggest estrogen-mediated effects on inflammation contributing 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 estrogen receptor– and PPAR-signaling pathways.47,48 Moreover, a few studies have shown gender-dependent responses to PPAR agonists.49,50 Nevertheless, relatively few patients were studied, the follow-up time was rather short, and our results from the psoriasis study should be interpreted with caution.
In the present study, we found that whereas TTA markedly enhanced PPAR-–target genes in the liver of wild-type, but not of PPAR-–/–, mice, no such effect on PPAR-–target genes was seen in HUVECs. The reasons for these different responses in the 2 model systems are at present unclear, but several reports suggest that these genes could be regulated differently in different organ systems and cell types. Thus, in mice, the expression of CD36 is regulated by PPAR- in the liver and by PPAR- in the adipose tissues.51 Moreover, we have previously reported that TTA can activate all PPAR subtypes,18,20,21 and, in particular, the ability of TTA to activate PPAR- may be of relevance for its antiinflammatory effects in endothelial cells.52 However, PPAR-independent mechanisms may also be operating. Indeed, we have shown antioxidant effects of TTA that, at least partly, may be PPAR independent, and such effects could clearly be of relevance in the modulation of TNF-–activated HUVECs at least partly through inhibition of nuclear factor B activation.53,54 Nevertheless, our findings underscore that the antiinflammatory effects of TTA seem to involve both PPAR-–dependent and –independent pathways, at least in some degree differing between the different model systems.
In the present study, we used different model systems to explore the potential immunomodulatory effects of TTA. Although the results cannot necessarily be extrapolated from 1 tissue to another, we believe that such an approach will also have some advantages. In fact, 1 of our main messages is that the effect of TTA, as well as other PPAR agonists, may differ among different tissues, underscoring the complexity when using such a therapeutic approach in human disorders. Nevertheless, our findings suggest an antiinflammatory potential of TTA that should be further investigated in disorders characterized by persistent inflammation and endothelial cell activation. In addition to therapeutic trials, such studies will also have to further clarify the mechanisms of action of TTA in inflammation.
Acknowledgments
This work was supported by grants from the Norwegian Association of Heart and Lung Patients, the University of Bergen, and the Inge Marie Larsine and Gabriel Tidemand Gabrielsens Legacy. We appreciate the use of equipment from the FFS-Medical Research Centre.
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