Published online before print November 21, 2007, doi: 10.1161/ATVBAHA.107.150250
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© 2008 American Heart Association, Inc.
Cell Biology/Signaling |
Activation of Thromboxane Receptor Upregulates Interleukin (IL)-1β–Induced VCAM-1 Expression Through JNK Signaling
From the Whitaker Cardiovascular Institute, Vascular Biology Unit, Department of Medicine, Boston University School of Medicine, Mass.
Correspondence to Bingbing Jiang, PhD, Vascular Biology Unit, X704, 650 Albany Street, Boston, MA 02118. E-mail bjiang{at}bu.edu
Abstract
Objective— Activation of thromboxane receptors (TPr) is implicated in atherosclerosis and inflammation. This study examined how activation of TPr modulates IL-1β–induced vascular cell adhesion molecule (VCAM)-1 expression in aortic vascular smooth muscle cells (VSMCs).
Methods and Results— In VSMCs, activation of TPr with U46619, a stable thromboxane A2 mimetic, alone did not induce VCAM-1 expression, but enhanced that caused by IL-1β. The enhancement of VCAM-1 expression caused by U46619 occurred at the transcriptional level and was inhibited either by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, or by overexpression of a dominant-negative JNK1, but not by SB203580, a p38 mitogen-activated protein kinase inhibitor. The activation of JNK by U46619 resulted in enhanced phosphorylation and nuclear translocation of c-Jun associated with an enhanced activation of activator protein (AP)-1, which were abolished by SQ29548, a TPr antagonist, or the JNK inhibitor. Treatment of the cells with U46619 alone did not induce NF-
B activation. Furthermore, U46619 enhanced IL-1β–induced THP-1 monocyte binding to VSMCs, which was inhibited by SQ29548 or SP600125.
Conclusion— This study demonstrates that activation of TPr upregulates IL-1β–induced VCAM-1 expression by enhancing the activation of JNK pathway that leads to enhanced AP-1 activation.
Activation of thromboxane receptors (TPr) is implicated in atherosclerosis and inflammation. This study demonstrated that in vascular smooth muscle cells, activation of TPr with U46619 alone does not induce vascular cell adhesion molecule (VCAM)-1 expression, but enhances IL-1β–induced VCAM-1 expression through enhancing JNK-dependent AP-1 activation, which enhances monocyte adhesion.
Key Words: adhesion molecules • cytokines • eicosanoids • receptors • smooth muscle cells
Atherosclerosis is a dynamic and progressive vascular disease associated with chronic vascular inflammation and increased oxidative stress.1,2 Increased formation of oxidized lipids derived from either low-density lipoproteins (LDL) or cell membrane phospholipids plays a critical role in the initial events of atherogenesis.3 A growing body of evidence suggests that many oxidized derivatives of arachidonic acid, including nonenzymatic peroxidation products, F2-isoprostanes, cyclooxygenase-produced prostaglandin (PG) H2 that is the precursor for production of thromboxane A2 and other prostaglandins, and lipoxygenase-produced hydroxyeicosatetraenoic acids, are involved in the vascular inflammatory process through activation of the thromboxane A2/PGH2 receptor (TPr).4,5 These arachidonic acid derivatives are elevated in patients with atherosclerotic vascular diseases, patients with diabetes mellitus, and animal models of atherosclerosis.6–14 Importantly, animal experiments have shown that blockade of TPr attenuates atherosclerosis in rabbits fed a high cholesterol diet, in nondiabetic and diabetic apolipoprotein E–deficient mice, and in LDL receptor–deficient mice,10–13 indicating that activation of TPr plays an important role in accelerating atherosclerosis.
We have previously shown that treatment with a TPr antagonist, S18886, attenuates expression of VCAM-1 and atherosclerotic lesions in the aorta of diabetic apolipoprotein E–deficient mice.11 This observation suggests that TPr are involved in the upregulation of VCAM-1 expression occurred during atherogenesis. VCAM-1 is a cell surface glycoprotein and its expression is enhanced in endothelial and intimal smooth muscle cells of atherosclerosis-prone regions of aorta. Together with other adhesion molecules and chemoattractants VCAM-1 mediates leukocyte adhesion to the endothelial cells and infiltration of the neointima.15–17 However, the interrelationship between TPr activation and the inflammatory response mediated by cytokines such as IL-1β has not been studied in aortic smooth muscle cells (VSMCs), and the mechanism by which blockade of TPr attenuates VCAM-1 expression is unknown. The present study aimed to determine how activation of the TPr with an agonist, U46619, enhances the expression of VCAM-1 induced by IL-1β in cultured VSMCs. We found that U46619 alone has no effect on nuclear factor (NF)-B activation and does not induce VCAM-1 expression. However, once NF-B activation is triggered by other stimuli such as IL-1β, U46619 enhances VCAM-1 expression through the c-jun N-terminal kinases (JNK) signaling pathway that leads to enhanced activation of activator protein (AP)-1.
Methods
Materials
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen. Recombinant human IL-β (specific activity: 1.9x107 U/mg) was kindly provided by Dr Aurigemma (the Biological Resources Branch Preclinical Repository, National Cancer Institute). U46619 (Biomol Research Laboratories), SP600125, and SB203580 (Calbiochem) were dissolved in dimethyl sulfoxide. SQ29548 was from Cayman Chemical. S18886 was obtained from Institut de Recherches Internationales Servier. Antibodies against VCAM-1 and heat shock protein (Hsp)-27 were from Santa Cruz Biotechnology. Antibodies against phospho-JNK (Thr183-Tyr185), JNK, phospho-c-Jun (Ser63), and phospho-Hsp27 (Ser82) were from Cell Signaling.
Cell Culture
Rat VSMCs, isolated from rat thoracic aorta of 8-week-old male Wistar rats,18 were cultured in DMEM with 10% FBS. The cells were used between passages 6 and 12. Human aortic VSMCs (ATCC, Catalog No: CRL-1999) were cultured in DMEM as described above, and used between passages 4 to 10. At confluence, the cells were rinsed with phosphate buffered saline (PBS) and maintained in DMEM with 0.1% FBS for 24 to 48 hours. The medium was refreshed before treatment. The cells were then incubated with the appropriate treatments as indicated. Unless specified otherwise, rat VSMCs were used in the experiments, IL-1β was routinely used at concentration of 3 ng/mL, U46619, SQ29548, and SP600125 were at 1 µmol/L, and SB203580 was at 10 µmol/L. Inhibitors were routinely added 1 hour before the addition of agonists. Human THP-1 monocytic cells (ATCC, Catalog No: TIB-202) were cultured in suspension in DMEM with 10% FBS. Coculture of the THP-1 monocytes with VSMCs is described below. The use of rats for preparation of VSMCs was approved by the Institutional Animal Care and Use Committee of Boston University Medical Center.
Adenoviral Constructs and Infection
Dominant-negative JNK1 (JNK1-dn, a mutant with T183A and Y185F) plasmid was generously provided by Dr Roger J. Davis (University of Massachusetts Medical School).19 Adenoviral construct was created using the Ad-Easy system (Qbiogen) as previously described.20 Adenovirus expressing β-galactosidase (LacZ) was used as a control. Confluent VSMCs cultured in 6-well plates were infected with the adenoviruses (4x1010 viral particles/mL) in DMEM with 0.1% FBS for 24 hours and the medium was refreshed before treatment for 24 hours with IL-1β in the absence or in the presence of U46619, as indicated.
Western Blot Analysis
Whole-cell lysates were prepared and Western blot analysis was performed as described previously.21 Protein content of the cell lysates were determined with BCA protein assay reagent (Pierce), with bovine serum albumin (BSA) used as a standard. Some experiments were performed on the Odyssey Licor Infrared Imaging System, where the secondary antibodies used were goat anti-rabbit IRDye 680 or goat anti-mouse IRDye 800 where appropriate.
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared and DNA-binding activities were assessed by electrophoretic mobility shift assay (EMSA) using [
-32P]-labeled either AP-1 consensus oligonucleotide (5'-CGC TTG AGT CAG CCG GAA-3'; Promega) or NF-B consensus oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3'; Promega) following the protocol described previously.21
Immunofluorescent Staining
To examine phospho-c-Jun nuclear translocation, VSMCs were cultured on 4-well Laboratory-Tek II chamber slides (Nalge Nunc International) under the same conditions described above. After treatment, the cells were washed with cold PBS, fixed for 8 minutes in methanol at –20°C, and air-dried at room temperature. The staining was performed by incubating with 10% normal goat serum in PBST for 60 minutes, followed by incubating with phospho-c-Jun polyclonal antibody (1 µg/mL) overnight at 4°C in PBST with 1.0% BSA, washing 3 times with PBS, incubating for 1 hour with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (1:100 dilution, Jackson ImmunoResearch Laboratories) in PBST with 1.0% BSA, washing 3 times with PBS, and finally mounting with aqueous mounting medium. The images observed under a fluorescence microscope were recorded on a linked computer with Openlab software (version 2.2.5, Improvision).
Luciferase Activity Assay
An adenoviral construct Ad.NFBluc (provided by Dr John Englehardt at University of Iowa Gene Transfer Vector Core) containing the luciferase reporter gene driven by 4 tandem copies of the NF-B consensus sequence fused to a TATA-like promoter from the herpes simplex virus-thymidine kinase gene22 was used in the luciferase assay. Confluent VSMCs cultured in 24-well plates were infected with the Ad.NFBluc (1.5x107 viral particles/mL) in DMEM with 0.1% FBS for overnight. The medium was refreshed before being incubated for 6 hours with the appropriate treatments as indicated. The cells were washed once with ice-cold PBS and then harvested in 150 µL cell lysis buffer (Stratagene). The cell lysates were centrifuged at 12 000g for 15 seconds at room temperature. Protein content of the lysate supernatants was determined with BCA protein assay reagent (Pierce). Equal volumes of luciferase substrate-assay buffer (Stratagene) and cell lysate supernatant were mixed together and after 8 seconds were placed into a 1251 Luminometer instrument for measurement of light with integration time of 2 seconds. The results are expressed as relative light units (RLU) per µg protein.
RT-PCR
VSMCs were cultured in 100-mm Petri dishes as described above. Total RNA was extracted from cells using TRIzol reagent (Invitrogen). The concentration of RNA was determined from the absorbance at 260 nm. The first-strand cDNA was synthesized from 1 µg total RNA by using random 9-mer and AMV-reverse transcriptase (Takara). PCR was performed with synthetic gene-specific primers for rat VCAM-1: forward 21-mer, 5'-ACA CCT CCC CCA AGA ATA CAG-3', and reverse 21 mer, 5'-GCT CAT CCT CAA CAC CCA CAG-3', which amplified a 477-bp sequence from +659 to +1135 of rat VCAM-1 cDNA according to the following schedule: denaturation, annealing, and extension at 95°C, 56°C, and 72°C for 40 seconds, 30 seconds, and 1 minute, respectively, for 28 cycles. A parallel PCR for GAPDH was performed as a reference with the same schedule and cycles as described above by use of a forward 20-mer, 5' GCC ATC AAC GAC CCC TTC AT-3', and reverse 20-mer, 5' CGC CTG CTT CAC CAC CTT CT-3', which amplified a 702-bp sequence from +88 to +789 of rat GAPDH cDNA. PCR products were electrophoresed on 1.2% agarose gels containing ethidium bromide, and visualized by UV-induced fluorescence.
Coculture
To examine THP-1 monocyte adhesion, VSMCs were cultured on 6-well plates under the same conditions described above. After treatment with IL-1β in the absence or in the presence of U46619, THP-1 monocytes were directly added to VSMCs and cocultured in 37°C incubator for 3 hours. The medium was then removed by aspiration and the plates were washed with PBS for 3 times to remove all nonbound THP-1 monocytes. Microscopic images were recorded on a linked computer, and bound THP-1 monocytes were counted and expressed as numbers per image area.
Statistical Analysis
Statistical analysis was performed using 1-way analysis of variance and Student t test. Results were expressed as means±SD. P<0.05 was considered significant.
Results
Activation of TPr With U46619 Upregulates IL-1β–Induced VCAM-1 Expression, Which Requires Activation of JNK
U46619 is an analog of PGH2 and a stable thromboxane A2 mimetic known to activate TPr.4 As shown in Figure 1A, treatment of rat VSMCs for 24 hours with IL-1β increased VCAM-1 expression. U46619 (0.01 to 1 µmol/L) enhanced IL-1β–induced VCAM-1 expression in a dose-dependent manner. However, treatment of the cells with U46619 alone did not induce VCAM-1 expression. The maximally effective dose of IL-1β for induction of VCAM-1 expression was about 10 ng/mL (supplemental Figure I, available online at http://atvb.ahajournals.org). The induction of VCAM-1 by IL-1β at either lower dose (3 ng/mL) or higher dose (25 ng/mL) was further enhanced by the presence of U46619 (supplemental Figure II). The effect of U46619 on upregulating IL-1β-induced VCAM-1 was completely abolished by blockade of TPr with either SQ29548 (Figure 1B), or S18886 (supplemental Figure III), both being highly selective antagonists of TPr. The enhancement by U46619 of IL-1β–induced VCAM-1 expression was also observed in cultured human VSMCs and was blocked by SQ29548 (Figure 1C). SQ29548 had no effect on VCAM-1 expression induced by IL-1β alone (Figure 1D), indicating that the induction of VCAM-1 by IL-1β is independent of TPr. These results indicate that activation of TPr alone cannot induce VCAM-1 expression, but can enhance IL-1β-induced VCAM-1 expression.
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Stimulation of TPr may induce the activation of mitogen-activated protein kinases (MAPKs), including extracellular signal regulated kinases (ERK) and JNK.23 In rat VSMCs, ERK activity is not required for VCAM-1 expression.20,24 We therefore examined whether inhibition of JNK with SP600125 or inhibition of p38 MAPK with SB203580 may influence the effect of U46619 on upregulating IL-1β induction of VCAM-1. The results shown in Figure 1B indicate that SP600125 prevented the enhancement by U46619 of IL-1β–induced VCAM-1 expression, whereas SB203580 had no significant effect. The specific role of JNK activation in TPr augmenting VCAM-1 expression by IL-1β was further investigated with adenoviral overexpression of a dominant-negative JNK1 (Figure 1E). The induction of VCAM-1 by IL-1β was significantly reduced and the enhancement of IL-1β–induced VCAM-1 by U46619 was abolished in the cells overexpressing JNK1-dn, whereas the induction was not affected by overexpressing Lac Z. These results indicate that activation of JNK is required for U46619 to enhance IL-1β–induced VCAM-1 expression.
U46619 Induces c-Jun Phosphorylation and Nuclear Translocation
To examine the role of TPr in activation of JNK and p38 MAPK pathways, a time course of U46619-induced phosphorylation of JNK and c-Jun, as well as the phosphorylation of Hsp27, a downstream target of p38 MAPK signaling, were examined by Western blot analysis (Figure 2). Upon treatment of the cells with U46619, phosphorylation of JNK was detected as early as 5 minutes, peaked at 10 to 15 minutes, and then rapidly decreased by 30 minutes, but was sustained at a lower level that was still detectable for up to 6 hours. The phosphorylation of c-Jun induced by U46619 was evident at 15 minutes, peaked at 1 hour, and was sustained for up to 6 hours. U46619 also induced Hsp27 phosphorylation in a time-dependent manner similar to that of phosphorylation of c-Jun, suggesting that stimulation of TPr also activates p38 MAPK.
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To confirm the specific inhibition of the JNK and p38 MAPK pathways by the selective inhibitors, we examined the phosphorylation of c-Jun and Hsp27, the downstream targets of JNK and p38 MAPK pathways, respectively, in the cells treated for 1 hour with IL-1β or U46619 or both. As shown in Figure 3A, either IL-1β or U46619 alone induced phosphorylation of c-Jun, however in combination of IL-1β and U46619, the phosphorylation of c-Jun was significantly enhanced. The phosphorylation of c-Jun was significantly inhibited by SP600125. IL-1β alone weakly induced the phosphorylation of Hsp27, consistent with our previous report that IL-1β has no or little effect on inducing p38 MAPK activation in rat VSMCs under our culture conditions.18,24 U46619 augmented Hsp27 phosphorylation, which was not prevented by the JNK inhibitor, SP600125. However, U46619-induced Hsp27 phosphorylation was prevented by the p38 MAPK inhibitor, SB203580 (Figure 3B). SB203580 did not inhibit c-Jun phosphorylation. The TPr antagonist SQ29548 abolished the ability of U46619 to induce the phosphorylation of both c-Jun and Hsp27. These results confirm the specificity of the inhibitors and are consistent with the observation that the augmentation of JNK but not p38 MAPK activity is required for U46619 to upregulate IL-1β–induced VCAM-1 expression.
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To determine whether IL-1β or U46619 causes translocation of phosphorylated c-Jun into the nucleus, a step that is important for the activation of the nuclear transcription factor AP-1, VSMCs were treated for 1 hour with IL-1β in the absence or presence of the TPr agonist, and the location of phosphorylated c-Jun was detected by immunofluorescent staining (Figure 3C). Fluorescence microscopy clearly shows the nuclear accumulation of phosphorylated c-Jun after treatment with either IL-1β or U46619 alone. The combination of IL-1β and the TPr agonist markedly enhanced the phosphorylated c-Jun in the nucleus. Figure 3C bottom shows the changes of relative stain intensities that were analyzed by the surface plot scan program of ImageJ (version 1.38a).
U46619 Enhances AP-1 Activation but not NF-B Activation
Because both NF-B and AP-1 are involved in activation of VCAM-1 transcription, we examined whether activation of TPr may activate these 2 transcription factors. Consistent with the phosphorylation and nuclear translocation of c-Jun, IL-1β alone or U46619 alone increased AP-1 DNA-binding activity as shown by EMSA, and the combination of IL-1β and U46619 further enhanced the DNA-binding activity of AP-1 (Figure 4A and 4B, left). The TPr antagonist, SQ29548, or JNK inhibitor, SP600125, dramatically attenuated IL-1β and U46619-induced AP-1 activation. As shown in Figure 4A and 4B (right), NF-B-DNA binding activity was observed in the nuclear extracts from the cells treated with IL-1β alone, but not in those treated with U46619 alone. NF-B DNA-binding activity induced by IL-1β plus U46619 was similar to that induced by IL-1β alone, and was not significantly influenced by the presence of either TPr antagonist SQ29548 or JNK inhibitor SP600125. NF-B–driven reporter gene luciferase activity assay also showed that U46619 alone did not trigger the reporter gene expression, whereas either IL-1β alone or IL-1β plus U46619 significantly induced luciferase activities (Figure 4C). These results demonstrate that stimulation of TPr alone does not activate NF-B, but activates AP-1, and can further enhance AP-1 activation in combination with IL-1β.
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U46619 Enhances VCAM-1 Transcription and THP-1 Monocyte Adhesion Induced by IL-1β
RT-PCR results shown in Figure 5A indicate that IL-1β–induced VCAM-1 transcription was enhanced by U46619. The effect of U46619 in upregulating IL-1β–induced VCAM-1 transcription was abolished by blockade of TP receptors with SQ29548. The JNK inhibitor, SP600125, also significantly reduced the mRNA levels of VCAM-1 in the cells treated with IL-1β plus U46619.
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To examine whether an increase in VCAM-1 expression in VSMCs may lead to an increase in monocyte adhesion, VSMCs were treated for 16 hours with IL-1β or U46619 or both. As shown in Figure 5B, VSMCs treated with IL-1β alone significantly increased the binding of THP-1 monocytes in comparison to untreated VSMCs (control). U46619 significantly enhanced IL-1β–induced THP-1 cell adhesion, although treatment of VSMCs with U46619 alone had no effect. In the presence of either SQ29548 or SP600125, the enhancement of IL-1β-induced adhesion of THP-1 cells to vascular smooth muscle cell (VSMC) by U46619 was prevented.
Discussion
In this study, we show that TPr stimulation of both rat and human VSMCs potentiates cytokine-induced VCAM-1 expression, providing a potential reason why activation of TPr potentiates development of vascular disease.10,11 In addition, we identified the signaling pathway by which TPr activation enhances VCAM-1 expression. Our data indicate that stimulation of TPr enhances IL-1β–induced VCAM-1 expression through augmented activation of JNK signaling that leads to increased AP-1 activation and VCAM-1 gene transcription that could be one of the factors leading to an increase in monocyte adhesion to smooth muscle cells.
Activation of both AP-1 and NF-B contributes to cytokine-induced VCAM-1 expression.20,25,26 In cultured endothelial cells, addition of TPr agonist alone causes VCAM-1 expression,27 probably because of the existence of IL-1β that is spontaneously produced by the cultured endothelial cells.28 Although it has been reported that either inhibition of NF-B by pyrolidine-dithiocarbamate or inhibition of both NF-B and AP-1 by a nonspecific inhibitor, N-acetylcysteine, suppressed the TPr agonist-induced VCAM-1 expression in the endothelial cells,27 the signaling mechanism responsible for enhanced VCAM-1 expression by activation of TPr in endothelial cells remains elusive. In the present study, we demonstrated that in cultured VSMCs, although U46619 alone induces JNK activation that leads to c-Jun phosphorylation and AP-1 activation, it is not sufficient to induce VCAM-1 expression. This is consistent with the fact that stimulation of TPr alone cannot induce NF-B activation in cultured VSMCs as shown by both EMSA and luciferase activity assay (Figure 4), and that activation of NF-B is essential for cytokine induction of VCAM-1.20 These results can further be generalized to other G protein–coupled receptors such as angiotensin II type-1 receptors. Like TPr, stimulation of angiotensin II type 1 receptors activates the JNK pathway but alone does not activate NF-B or induce VCAM-1, while it does enhance IL-1β–induced VCAM-1 expression in rat VSMCs.20
AP-1 consists of homo- and heterodimers that are composed of the basic region-leucine zipper proteins belonging to the Jun (c-Jun, v-Jun, JunB, JunD) and Fos (c-Fos, v-Fos, FosB, Fra1, Fra2) subfamilies.29 Regulation of AP-1 activity occurs by activating transcription of these genes as well as through phosphorylation of existing Jun and Fos proteins at specific serine and threonine sites. The present study demonstrated that phosphorylated c-Jun was increased in the nucleus in VSMCs treated with either IL-1β or U46619 alone, and was greatly enhanced by their combination. The phosphorylation and nuclear accumulation of c-Jun is consistent with the activation of AP-1 by either IL-1β or U46619, or both. Both TPr antagonist SQ29548 and JNK inhibitor SP600125 effectively inhibited TPr activation–induced c-Jun phosphorylation and AP-1 DNA-binding activity, which is consistent with their inhibition of the TPr-mediated enhancement of VCAM-1 expression. Treatment of VSMCs with U46619 also activated p38 MAPK. However, activation of p38 MAPK was unlikely to contribute to the role of U46619 in enhancing IL-1β–induced VCAM-1 expression, because inhibition of p38 MAPK activity by SB203580 showed no effect. Protein kinase C may also mediate TPr signaling in endothelial cell expression of adhesion molecules27 but the mechanism is unknown. The diagram presented in Figure 6 summarizes the mechanism by which TPr activation enhances IL-1β–induced VCAM-1 expression and monocyte adhesion.
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VCAM-1 is expressed in both endothelial cells and intimal smooth muscle cells in atherosclerotic lesions in humans and in animal models, and is associated with the recruitment of leukocytes and vascular inflammation.30–34 In atherosclerosis-prone regions of the aorta, the expression of VCAM-1 in endothelial cells is one of the early events observed in proatherosclerotic conditions such as hyperlipidemia and hyperglycemia. This results in leukocyte adhesion to, and transmigration of, the endothelium. Particularly in advanced atherosclerotic lesions in both human and animal arteries, VCAM-1 expression is also frequently detected in smooth muscle cells in intimal lesions and in the media adjacent to the lesions31,32,34 (also see supplemental Figure IV). In coculture studies, we demonstrated that IL-1β increased THP-1 monocyte binding to VSMCs, which was augmented by TPr activation. However, activation of TPr alone had no effect on monocyte binding to VSMCs. The increase in monocyte adhesion is correlated with the increase in VCAM-1 expression in our model. VCAM-1 expressed in smooth muscle cells may facilitate the accumulation of transmigrated leukocytes within the intima during the development of atherosclerosis. The adhesion of monocytes to VSMCs upregulates monocyte expression of CD36, a class B scavenger receptor, and this step, which is critical for differentiation into macrophage foam cells can be blocked by VCAM-1 neutralizing antibody.17
Blockade of TPr attenuates atherosclerosis in both nondiabetic and diabetic animals with hyperlipidemia.10–13 However, treatment with TPr antagonists in those animals did not show any significant improvement in their plasma lipid levels and glucose levels. Based on the evidence from both in vivo and in vitro experiments, and our experiments performed on human VSMCs, we postulate that an important mechanism by which blockade of TPr attenuates atherosclerosis is by abolishing the enhancement of VCAM-1 expression by TPr agonists including eicosanoids produced enzymatically and isoprostanes produced nonenzymatically from arachidonic acid. Taken together, inhibition of TPr-mediated JNK activation leading to c-Jun translocation, AP-1 activation, and enhanced VCAM-1 expression may represent an important therapeutic target in reducing vascular inflammation and atherogenesis.
Acknowledgments
Sources of Funding
This study was supported in part by an American Heart Association (AHA) grant 0435205N, National Institute of Health (NIH) grants R01 HL55620, R01 AG27080, P01 HL68758, and a strategic alliance between the Vascular Biology Unit at Boston University Medical Center and the Institut de Recherche Servier.
Disclosures
None.
Footnotes
Original received November 28, 2006; final version accepted September 7, 2007.
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