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The Peroxisome Proliferator-Activated Receptor- Agonist Pioglitazone Represses Inflammation in a Peroxisome Proliferator-Activated Receptor-–Dependent Manner In Vitro and In Vivo in Mice

Gabriela Orasanu, MD^_*,, Ouliana Ziouzenkova, PhD^_*,, Pallavi R. Devchand, PhD^_*,, Vedika Nehra, MS^_*,, Osama Hamdy, MD^,, Edward S. Horton, MD^, and Jorge Plutzky, MD^_*,,_*

^_* Cardiovascular Division, Brigham and Women's Hospital
Clinical Research Center, Joslin Diabetes Center
Harvard Medical School, Boston, Massachusetts. Dr. Plutzky has received grant support from the National Institutes of Health (R01 HL071745)

Manuscript received March 10, 2008; revised manuscript received April 22, 2008, accepted April 29, 2008.

^_* Reprint requests and correspondence: Dr. Jorge Plutzky, Brigham and Women's Hospital, 77 Avenue Louis Pasteur, NRB 742, Boston, Massachusetts 02115 (Email: jplutzky{at}rics.bwh.harvard.edu).

	Abstract

Top Abstract Methods Results Discussion Appendix References

 
Objectives: Our aim was to investigate if the peroxisome proliferator-activated receptor (PPAR)- agonist pioglitazone modulates inflammation through PPAR mechanisms.

Background: The thiazolidinediones (TZDs) pioglitazone and rosiglitazone are insulin-sensitizing PPAR agonists used to treat type 2 diabetes (T2DM). Despite evidence for TZDs limiting inflammation and atherosclerosis, questions exist regarding differential responses to TZDs. In a double-blinded, placebo-controlled 16-week trial among recently diagnosed T2DM subjects (n = 34), pioglitazone-treated subjects manifested lower triglycerides and lacked the increase in soluble vascular cell adhesion molecules (sVCAM)-1 evident in the placebo group. Previously we reported PPAR but not PPAR agonists could repress VCAM-1 expression. Since both triglyceride-lowering and VCAM-1 repression characterize PPAR activation, we studied pioglitazone's effects via PPAR.

Methods: Pioglitazone effects on known PPAR responses—ligand binding domain activation and PPAR target gene expression—were tested in vitro and in vivo, including in wild-type and PPAR-deficient cells and mice, and compared with the effects of other PPAR (rosiglitazone) and PPAR (WY14643) agonists.

Results: Pioglitazone repressed endothelial TNF-induced VCAM-1 messenger ribonucleic acid expression and promoter activity, and induced hepatic IB in a manner dependent on both pioglitazone exposure and PPAR expression. Pioglitazone also activated the PPAR ligand binding domain and induced PPAR target gene expression, with in vitro effects that were most pronounced in endothelial cells. In vivo, pioglitazone administration modulated sVCAM-1 levels and IB expression in wild-type but not PPAR-deficient mice.

Conclusions: Pioglitazone regulates inflammatory target genes in hepatic (IB) and endothelial (VCAM-1) settings in a PPAR-dependent manner. These data offer novel mechanisms that may underlie distinct TZD responses.

Key Words: inflammation • VCAM-1 • PPAR-s

Abbreviations and Acronyms   ACO = acyl-CoA oxidase   BAEC = bovine aortic endothelial cells   EC = endothelial cell   FPG = fasting plasma glucose   GAPDH = glyceraldehyde-3-phosphate dehydrogenase   HbA1c = hemoglobin A1c   HDL = high-density lipoprotein   HSVEC = human endothelial cells isolated from saphenous vein   LBD = ligand binding domain   LDL = low-density lipoprotein   LPL = lipoprotein lipase   LPS = lipopolysacharide   PPAR = peroxisome proliferator-activated receptor   TG = triglyceride(s)   TNF = tumor necrosis factor   TZD = thiazolidinedione   T2DM = type 2 diabetes mellitus   VCAM = vascular cell adhesion molecule   2h-OGTT = 2-h plasma glucose 200 mg/dl during oral glucose tolerance testing

TZDs bind to and activate PPAR, a ligand-activated transcription factor that regulates key metabolic pathways, including adipogenesis and insulin sensitivity (6,7). Pioglitazone and rosiglitazone are approved as "highly selective" peroxisome proliferator-activated receptor (PPAR)- agonists (8). PPAR is also expressed in vascular and inflammatory cells where its activation can regulate target genes relevant to atherosclerosis (9). Although pioglitazone and rosiglitazone target the same PPAR isoform (4), recent human TZD data have raised the possibility of variable responses between these agents as well as with other PPAR agonists. In several studies, including one head-to-head clinical trial, pioglitazone and rosiglitazone have had variable as well as divergent effects on triglycerides (TG) (10,11). Meta-analysis data have suggested possible adverse cardiovascular outcomes with rosiglitazone (12–14), although not without controversy (15,16); similar studies with pioglitazone have not shown cardiovascular safety signals, including one prospective clinical trial (13,17). Novel dual PPAR/ agonists in development have been abandoned for various adverse effects, including cardiovascular responses (12), raising concerns about dual PPAR/ therapeutics (18,19). Clearly, additional mechanistic insight into how specific PPAR- agonists exert their effects is needed.

During a small 16-week study on inflammatory markers in patients with recently diagnosed T2DM randomized to pioglitazone or placebo, we noted that levels of soluble vascular cell adhesion molecule (sVCAM)-1, an early player in atherogenesis, increased in the placebo group but not in the group on pioglitazone (20). Previously we reported that PPAR activation had no effect on VCAM-1 expression in vitro, although those studies did not include pioglitazone (21). In contrast, PPAR agonists decrease VCAM-1 in a PPAR-dependent manner (22). Although limited in nature, this clinical data raised the hypothesis that pioglitazone might repress inflammation, including VCAM-1 expression, in part via PPAR. Although previously raised as a possibility, we studied pioglitazone effects on known PPAR responses in more definitive models, including PPAR-deficient cells and mice (23). We provide here the first evidence that pioglitazone represses key endothelial and hepatic inflammatory responses in vitro and in vivo in mice in a PPAR-dependent manner.

	Methods

Top Abstract Methods Results Discussion Appendix References

 
Human studies.   Subjects
Individuals meeting the American Diabetes Association criteria for T2DM—fasting plasma glucose (FPG) 126 mg/dl with a second confirmatory measurement or a 2-h plasma glucose 200 mg/dl during oral glucose tolerance testing (2h-OGTT)—were enrolled in a randomized, prospective, double-blinded, placebo-controlled clinical trial (n = 34). The study cohort included subjects with newly diagnosed T2DM or confirmed T2DM on a nonpharmacological dietary intervention for at least 4 weeks before the first study visit; all subjects were either drug-naïve or off any antidiabetic medication for at least 4 weeks. Exclusion criteria included prior TZD or insulin treatment, hemoglobin A_1c (HbA_1c) 10%; FPG 260 mg/dl; history of myocardial infarction, unstable angina, cerebral vascular accident, transient ischemic attack, coronary artery bypass graft, or percutaneous transluminal coronary angioplasty; New York Heart Association functional class III or IV congestive heart failure; diastolic blood pressure >100 mm Hg and/or systolic blood pressure >160 mm Hg; total cholesterol >300 mg/dl and/or TG >600 mg/dl; serum creatinine >1.5 mg/dl; current use of systemic corticosteroids, immunosuppressants, or androgens; any severe acute or chronic disease; other medical condition possibly interfering with study participation or assessment of the trial investigational products.

Protocol
Participants, recruited from the Joslin Diabetes Center outpatient clinics or local media advertisement, were randomized 1:1 to either pioglitazone 30 mg tablet or a matching placebo once a day for the first 4 weeks, with a subsequent increase to pioglitazone 45 mg tablet or matched placebo once a day for the next 12 weeks (total 16 week intervention period). Both groups were evaluated at baseline and study conclusion. Patients were instructed on an isocaloric diet (50% carbohydrates, 20% protein, 30% fat) and to maintain their usual physical activity.

Laboratory evaluations
All measurements were performed at the Clinical Research Center, Joslin Diabetes Center (24). Plasma sVCAM-1 concentrations in mice and humans were determined in duplicate blinded samples using ELISA (Quantikine, R&D Systems, Minneapolis, Minnesota). Intra-assay variation was less than 10%; the sVCAM-1 detection limit was 0.6 ng/ml. To analyze sVCAM-1 responses in different subgroups, national guideline cutpoint values were used: above and below FPG 126 mg/dl, 2h-OGTT 200 mg/dl, HbA_1c 7%, TG 150 mg/dl, low-density lipoprotein (LDL) cholesterol 100 mg/dl, high-density lipoprotein (HDL) cholesterol 40 mg/dl (25,26).

Reagents.   Pioglitazone hydrochloride (pioglitazone HCl) was a gift from Takeda Pharmaceuticals North America (Lincolnshire, Illinois); WY14643 (Biomol, Plymouth Meeting, Pennsylvania); rosiglitazone (BRL49653, GlaxoSmithkline, Research Triangle Park, North Carolina). All media (BioWhittaker, Walkersville, Maryland) contained fungizone, penicillin, streptomycin, and plasma as indicated. Human and murine TNF were purchased from R&D Systems; Escherichia coli O111:B4 lipopolysacharide (LPS) and 2, 2, 2-tribromoethanol (Sigma-Aldrich, St. Louis, Missouri).

Cell culture.   Human endothelial cells isolated from saphenous veins (HSVECs) were cultured in M199 medium, endothelial cell (EC) growth factor, and 5% fetal calf serum (21). Bovine aortic endothelial cells (BAECs) were grown in Dulbecco modified Eagle medium (10% fetal bovine serum, glutamine, penicillin, streptomycin, and fungizone) (27). Tumor necrosis factor (TNF) stimulations were done at 10 ng/ml. PPAR^+/+ (129S1/SvImJ) mice were obtained from Jackson Laboratories (Bar Harbor, Maine). PPAR^–/– mice (129S4/SvJae) were a generous gift from F. Gonzalez (National Institutes of Health). Murine ECs from 1-month-old PPAR^+/+ and -^–/– mouse hearts were isolated using double selection with intercellular adhesion molecule 2 and platelet EC adhesion molecule 1 antibodies (BD PharMingen, San Diego, California) bound to Dynabeads (Dynal, Lake Success, New York) as before (28).

Plasmids.   Human GAL4-PPAR- or -–ligand binding domain (LBD) constructs (pSG5 vector, S. Kliewer, University of Texas Southwestern, Dallas, Texas) were used for transactivation assays. The VCAM-1 promoter construct (755 upstream base pair [bp], T. Collins, Children's Hospital, Boston, Massachusetts) contains the major regulatory elements (AP-1, NF-B, PPAR) (21). cDNA probes for Northern blotting included human VCAM-1 (2.1kb Kpn/Sphl fragment, G. Garcia-Cardena, Brigham and Women's Hospital, Boston, Massachusetts); mouse full-length PPAR (2kb cDNA fragment); human acyl-CoA-oxidase (ACO; ATCC, Manassas, Virginia); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ATCC).

Transient transfection assays.   Standard GAL4-PPAR–LBD assays were performed as before (24-well plates, 2.5 x 10⁴ BAECs/well, passage 2 to 5, FuGENE 6 (Roche Diagnostics, Indianapolis, Indiana) (27) in Dulbecco modified Eagle medium (1% delipidated fetal calf serum) using human GAL4-PPAR or --LBD, pUASx4-TK-luc, and β-galactosidase (pcDNA-β-Gal) constructs before stimulation (24 h later) with the compounds indicated for 16 h. For VCAM-1 promoter studies, BAECs were plated in 1% Nutridoma SP (Roche Diagnostics), transfected with the VCAM-1 promoter construct (24 h), and then pre-treated with pioglitazone (3 to 30 µM) or WY14643 (25 to 225 µM, 3 h) before TNF stimulation (12 h). Responses were normalized to co-transfected β-galactosidase (pcDNA3) activity using chlorophenol red-β-D-galactopyranoside substrate (Roche Diagnostics) as before (27). For PPAR reconstitution experiments, murine PPAR^–/– ECs were plated in 1% delipidated fetal calf serum before transfection with either PPAR (mouse full-length PPAR-pSG5) or empty vector (pSG5, 24 h). Cells were pre-treated with pioglitazone (10 µM) or WY14643 (100 µM) for 18 h before mouse TNF (10 h) stimulation as indicated.

Ribonucleic acid extraction and Northern blot analysis..   Total ribonucleic acid was isolated using RNeasy (Qiagen, Valencia, California) before gel separation and transfer (Hybond-N, Amersham Pharmacia, Piscataway, New Jersey). Northern and Western blots were quantitated using densitometry (Image-Pro Plus 5.1 software).

Western blotting.   Standard Western blot analysis of human EC lysates were performed using rabbit polyclonal antibody against human IB (1:500, Santa Cruz Biotechnology, Santa Cruz, California) and monoclonal antibody against GAPDH (1:10,000, Biodesign, Saco, Maine). For in-vivo studies, frozen livers were pulverized, added to RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) with freshly added protease inhibitors, centrifuged (10', 13,000 rpm, 4°C), and the protein extract boiled in electrophoresis buffer before gel separation (15% polyacrylamide, β-mercaptoethanol-reducing conditions) and transfer (Immobilon-P membranes, semi-dry transfer, 1 h, 16 V). After blocking (5% delipidated milk, 20 mM Tris, 55 mM NaCl, 0.1% Tween 20, 1 h), immunoblotting with the IB and GAPDH antibodies described in the previous text was performed using chemiluminescence (PerkinElmer Life Sciences, Boston, Massachusetts).

Animal studies.   The 3-month-old age- and gender-matched PPAR^+/+ and PPAR^–/– mice were divided into 2 feeding groups (n = 9/genotype). Group One received pioglitazone (20 mg/kg body weight in 0.5% w/v methylcellulose) by gavage once daily for 7 days. Group Two was treated similarly with vehicle (0.5% w/v methylcellulose). Mice received free access to water, ordinary laboratory diet, and standard animal care (Harvard University guidelines). On Day 1, mice received a survival dose of anesthetic (2, 2, 2—tribromoethanol, 250 mg/kg body weight) intraperitoneally before retro-orbital blood draw for baseline serum measurements. On Day 8, retro-orbital blood draws were repeated on a random subgroup of mice (n = 5). The next day, all mice received LPS (12 mg/kg body weight) intraperitoneally; 4 h later, mice were anesthetized and blood drawn by vena cava puncture and allowed to clot overnight (4°C). Mice were then sacrificed and livers removed immediately, rinsed (0.9% NaCl), and snap-frozen for further analysis. Blood samples were centrifuged (2,000 x g, 4°C, 20') and serum sVCAM-1 concentrations determined in duplicate as described in the preceding text.

Statistical analysis.   Statistical analysis, performed in conjunction with the Brigham and Women's Hospital Center for Clinical Research Biostatistics Core Laboratory, employed Statistical Package for Social Sciences (version 16.0, SPSS Inc., Chicago, Illinois), SAS 9.1 (SAS Institute, Cary, North Carolina), and Analyze-it for Microsoft Excel (version 1.71). Results are presented as mean ± SD or mean ± SE. Means for baseline clinical characteristics of the human study participants were compared using the independent Student t test. For within-group analysis (the baseline study vs. the follow-up assessment), 2-sided paired Student t test for parametric data was used. For all other among-group comparisons, the Mann-Whitney U test was used. Pearson correlation coefficients were calculated to test the association between variables. A value of p 0.05 was regarded as significant.

	Results

Top Abstract Methods Results Discussion Appendix References

 
Changes in sVCAM-1 on pioglitazone versus placebo in recently diagnosed T2DM subjects.   Pioglitazone (n = 19) and placebo (n = 15) groups were similarly matched on all baseline variables, including sVCAM-1 levels (Table 1). Pioglitazone significantly improved FPG (162.2 ± 13.6 vs. 125.4 ± 7.1 mg/dl, p = 0.002), 2h-OGTT (273.5 ± 19 vs. 216.3 ± 12.6, p = 0.001), TG (160.7 ± 24.9 vs. 129.1 ± 11.4, p = 0.008), and TG/HDL ratio (3.5 ± 0.5 vs. 3.1 ± 0.2, p = 0.02), all as compared with placebo at baseline versus study end (Table 1).

To generate hypotheses as to biologic mechanisms underlying possible pioglitazone effects on repressing the sVCAM-1 increase seen in patients on placebo, responses were analyzed according to the intervention arm and subgroups stratified by accepted TG, HDL cholesterol, LDL cholesterol, FPG, and HbA_1c cutpoints (see Methods section). Only TG subgroups revealed differences in sVCAM-1 levels. Using the National Cholesterol Education Program TG cutpoint of 150 mg/dl (29), significant sVCAM-1 increases were restricted to those with higher baseline TG levels (150 mg/dl, n = 9; from baseline 506 ± 63.9 ng/ml to 683.1 ± 56.4 ng/ml, p < 0.03); sVCAM-1 levels did not differ significantly in placebo-treated subjects with lower baseline TG (<150 mg/dl, n = 6). Among pioglitazone-treated subjects, sVCAM-1 levels did not differ in either higher or lower TG subgroups (data not shown).

Pioglitazone represses TNF-induced VCAM-1 messenger ribonucleic acid (mRNA) expression in ECs..   Given the data above, we tested pioglitazone's effects on TNF-induced VCAM-1 mRNA expression in HSVECs (18 h pre-treatment) using a concentration range commonly used in vascular biology studies which overlap pioglitazone levels reported in humans (30). Pioglitazone inhibited VCAM-1 mRNA induction in a dose-dependent manner (Fig. 1A). For comparison, the known repression of VCAM-1 mRNA by the PPAR agonist WY14643 (100 µM) is also shown (Fig. 1A). Quantification of relative changes in VCAM-1/GAPDH mRNA expression using densitometry reveals a significant pioglitazone effect at the concentrations shown (Fig. 1B) (3 to 30 µM, p < 0.05 for each). As previously reported, rosiglitazone (BRL49653, BRL) had no significant effect on VCAM-1 expression (data not shown) (21,31–33). Pioglitazone-mediated repression of VCAM-1 expression also varied as a function of pioglitazone exposure (3, 6, 18 h; 10 µM) before TNF stimulation (maximal 74% reduction at 18 h, p < 0.05) (Fig. 1C).

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Pioglitazone Reduces TNF-Induced VCAM-1 mRNA Expression in a Dose- and Time-Dependent Manner in HSVECs (A) Northern blot analysis of tumor necrosis factor (TNF)-induced vascular cell adhesion molecule (VCAM)-1 messenger ribonucleic acid (mRNA) expression was performed on human saphenous vein endothelial cells (HSVECs) pre-treated in the absence or presence of pioglitazone (PIO) (18 h) at the concentrations shown before TNF stimulation (10 ng/ml, 10 h). The effects of the PPAR agonist WY14643 (100 µM) are provided for comparison. One representative Northern blot (n = 3) is shown. (B) The effect of the pioglitazone concentrations on VCAM-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was quantified from the Northern blots seen in panel A (n = 3, #p < 0.05, TNF-induced vs. vehicle, *p < 0.05, pioglitazone/TNF vs. TNF alone, Mann-Whitney U test). (C) The time-dependent effects of pioglitazone exposure (10 µM) on TNF-induced VCAM-1 expression was tested in HSVECs using Northern blotting. Results are shown as a percent of the TNF effect alone at 3 h, mean ± SD (n = 3; *p < 0.05). (D) The effect of pioglitazone versus vehicle on the human VCAM-1 promoter transiently transfected into bovine aortic endothelial cells before TNF stimulation are shown (left). For comparison, the effect of the PPAR agonist WY14643 on the VCAM-1 promoter is also shown (right). All responses were normalized to β-galactosidase (pCMV-β-Gal) (n = 3 per each treatment, #p < 0.05 TNF vs. vehicle; *p < 0.05 pioglitazone or WY14643 vs. TNF alone, Mann-Whitney U test).

Pioglitazone regulation of VCAM-1 mRNA expression in the genetic presence or absence of PPAR.   We next considered if pioglitazone's effects on VCAM-1 required the genetic presence of PPAR. In Northern blots of microvascular ECs isolated from either wild-type (PPAR^+/+) or PPAR-deficient (PPAR^–/–) mouse hearts, WY14643 (100 µM) and pioglitazone (10 µM) pre-treatment decreased VCAM-1 mRNA expression in PPAR^+/+ but not in PPAR^–/– ECs while BRL (1 µM) had no effect in either PPAR^+/+ or PPAR^–/– ECs (Figs. 2A and 2B), as seen in Northern blotting. Pioglitazone significantly decreased TNF-induced VCAM-1 mRNA expression in a dose-dependent manner (3 to 30 µM, 18 h) in wild-type ECs (as compared with TNF stimulation alone, p < 0.05) (Fig. 2C) but not in PPAR^–/– ECs (Fig. 2D). Quantification of these responses (n = 3) demonstrated a significant VCAM-1 effect at each pioglitazone dose tested in wild-type but not PPAR-deficient ECs (p < 0.05) (Fig. 2E).

View larger version (47K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Pioglitazone Represses TNF-Induced VCAM-1 Expression in a PPAR-Dependent Manner Endothelial cells (ECs) isolated from PPAR+/+ (A) and PPAR–/– (B) mouse hearts were pre-treated with WY14643, rosiglitazone (BRL), or pioglitazone at the concentrations shown (18 h) before mouse TNF stimulation and subsequent to Northern blotting for VCAM-1 mRNA and GAPDH expression. One representative blot of 3 is shown. Northern blotting for VCAM-1 expression was repeated in the presence of the dose range of pioglitazone shown in EC from PPAR+/+ (C) and PPAR–/– (D) mice. (E) Quantification of the effects of pioglitazone on VCAM-1 mRNA in PPAR+/+ and PPAR–/– EC relative to GAPDH mRNA expression (n = 3, #p < 0.05 TNF vs. vehicle; *p < 0.05 pioglitazone/TNF vs. TNF alone, Mann-Whitney U test). Abbreviations as in Figure 1.

Reconstitution of PPAR expression in PPAR-deficient ECs.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 3 PPAR Is Required for Pioglitazone-Mediated Repression of TNF-Induced Endothelial VCAM-1 mRNA Expression Northern blot analysis was performed on total RNA isolated from PPAR–/– endothelial cells transfected either with PPAR-containing pSG5 overexpression vector (A) or pSG5 alone (B) before stimulation with TNF in either the absence or presence of WY (100 µM) or pioglitazone (10 µM), with subsequent probing for VCAM-1 or GAPDH expression. (C) Quantification of the VCAM-1 mRNA response to pioglitazone relative to GAPDH mRNA expression levels (n = 3, #p < 0.05 TNF vs. vehicle; *p < 0.05 pioglitazone/TNF vs. TNF alone; p < 0.05 WY14643/TNF vs. TNF alone, Mann-Whitney U test). (D) Cells were transfected in panels A and B but with a concentration gradient of pSG5-PPAR as shown before TNF stimulation in either the absence or presence of pioglitazone 10 µM (n = 3, *p < 0.05 pioglitazone/TNF vs. TNF alone). Abbreviations as in Figure 1.

Pioglitazone effects on expression of canonical PPAR-regulated target genes.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Pioglitazone Induces Known PPAR- Target Gene Expression and PPAR-LBD Activation in ECs (A) Northern blot analysis in HSVECs was performed for the PPAR target gene acyl-CoA-oxidase (ACO) and compared with GAPDH in HSVEC pre-treated (16 h) with pioglitazone or WY14643 at the concentrations shown before TNF stimulation. (B) Western blot analysis for IB expression was performed on total protein extracts (50 µg) from HSVECs treated with either pioglitazone (10 µM) or WY14643 (250 µM) before stimulation with human TNF. (C) Standard LBD activation assays were performed in bovine aortic endothelial cells stimulated with pioglitazone at the concentrations shown (0.01 to 100 µM). (D) PPAR-ligand binding domain (LBD) assays were done as before but responses were compared in NIH/3T3 (fibroblasts), HEK293 (human kidney epithelial), Hep-G2 (hepatic), and bovine aortic endothelial cell lines before stimulation with pioglitazone or WY14643 (both 10 µM). Values are expressed as luciferase/β-Gal activity mean ± SD (n = 3, *p < 0.05 bovine aortic endothelial cells vs. NIH/3T3, **vs. HEK293, ***vs. Hep-G2, both Student t and Mann-Whitney U tests). Abbreviations as in Figure 1.

Pioglitazone's PPAR-dependent effects on hepatic IB protein levels in vivo.   We next considered if pioglitazone regulated PPAR responses in a PPAR-dependent manner in vivo. Given the TG and sVCAM-1 effects shown here (Table 1), we focused on pioglitazone responses in tissues related to lipid metabolism (liver) and inflammation (endothelium), and relevant PPAR-regulated inflammatory target genes in those settings, namely IB and VCAM-1. PPAR^+/+ (n = 4) and PPAR^–/– (n = 4) mice were treated with pioglitazone (gavage, 20mg/kg body weight, 7 days) before harvesting livers and performing IB Western blotting. Consistent with our in vitro results, pioglitazone significantly increased hepatic IB protein expression in PPAR^+/+ (Fig. 5A) but not PPAR^–/– (Fig. 5B) mice, as evident on densitometry (p < 0.05) (Fig. 5C)

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Pioglitazone Induces IB Protein Expression In Vivo in a PPAR-Dependent Manner PPAR+/+ (A) and PPAR–/– (B) mice were treated with pioglitazone (20 mg/kg, 7 days via gavage) before livers were harvested and total protein extracted for Western blot analysis of IB and glyceraldehyde-3-phosphate dehydrogenase protein levels. Each lane represents a single mouse. (C) The effects of pioglitazone (solid bars) and vehicle (open bars) on IB protein expression were quantified and normalized to glyceraldehyde-3-phosphate dehydrogenase expression. Mean values ± SD are shown (n = 4 mice/group. *p < 0.05 pioglitazone vs. vehicle, Mann-Whitney U test).

Pioglitazone's PPAR-dependent effect on sVCAM-1 levels in vivo.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Pioglitazone Decreases LPS-Induced Soluble VCAM-1 in PPAR+/+ But Not PPAR–/– Mice In Vivo PPAR+/+ and PPAR–/– mice were treated with pioglitazone or vehicle alone before lipopolysacharide (LPS) injection (n = 9/genotype as in Methods section). Soluble vascular adhesion molecule-1 (sVCAM-1) levels in PPAR+/+ and PPAR–/– mice are shown at baseline (*p < 0.007 PPAR–/– vs. PPAR+/+ mice) and after LPS injection in mice treated with either vehicle or pioglitazone (PIO) (PPAR+/+, n = 9, #p < 0.002 LPS/vehicle vs. vehicle; p < 0.01 pioglitazone/LPS vs. vehicle/LPS) and in PPAR–/– mice (n = 9, p < 0.05 vehicle/LPS vs. vehicle; [p = NS] nonsignificant pioglitazone/LPS vs. vehicle/LPS, significance determined using Mann-Whitney U test). The mean serum sVCAM-1 concentration of each group ± SD is shown.

	Discussion

Top Abstract Methods Results Discussion Appendix References

Agonists for the same PPAR isoform can differ significantly in their biologic and clinical effects. In transcriptional profiling and proteomic assays, different PPAR agonists have both shared and distinct gene expression patterns (39–41). Clinically, both pioglitazone and rosiglitazone lack the irreversible liver failure seen with troglitazone, the first clinically approved PPAR agonist (42). In a head-to-head clinical trial, pioglitazone decreased TG significantly while rosiglitazone did not (11), as also suggested by meta-analysis data (43). Although the contribution of differential TZD effects to cardiovascular events remains unclear, our data identify an additional biologic mechanism that may be involved in pioglitazone responses. Considerable evidence establishes TZDs as limiting inflammation and atherosclerosis in mouse models (45). In clinical studies, both pioglitazone and rosiglitazone lower C-reactive protein (38,46–49), anatomic indicators like carotid intimal medial thickness (49–53), and vessel reactivity (54). In the prospective PROactive (PROspective pioglitAzone Clinical Trial In macroVascular Events) study, pioglitazone did not decrease a large, combined primary cardiovascular end point although a secondary clinical end point of stroke, myocardial infarction, and cardiovascular death was improved (55). Various logistical factors have been speculated as contributors to the study's negative primary end point (4). Recently, in the PERISCOPE (Pioglitazone Effect on Regression of Intravascular Sonographic Coronary Obstruction Prospective Evaluation) trial, progression of atherosclerosis was seen on coronary intravascular ultrasound in otherwise well-treated patients randomized to glimiperide but not in those receiving pioglitazone (56). Pioglitazone significantly decreased TG and raised HDL in both the PROactive and PERISCOPE trials. No large prospective clinical cardiovascular rosiglitazone trial data is currently available. Recent meta-analyses have raised concern over a possible increase in cardiovascular risk with rosiglitazone (12–14,55), although with limitations in this data as raised by the authors and others (15,55). In similar meta-analyses, no increased risk with pioglitazone was found (13). Given changes induced by TZDs on surrogate markers for cardiovascular disease in basic and clinical studies, it remains possible that offsetting adverse cardiovascular effects could exist with these agents. PPAR activation can increase homocysteine levels and serum creatinine levels (57–60). While the VA-HIT (Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial) study did show decreased cardiovascular events with the putative PPAR agonist gemfibrozil, in the FIELD (Fenofibrate Intervention and Event Lowering in Diabetes) study, fenofibrate, a more potent PPAR agonist, did not show a difference in the primary cardiovascular end point (61,62). Although the role of pioglitazone-mediated PPAR activation in determining clinical responses remains unclear, our data suggest at the very least that PPAR activation should be considered in interpreting basic science data with this agent.

Both VCAM-1 and IB regulate inflammatory responses in atherosclerosis. Endothelial VCAM-1 expression is an important early atherogenic step (2). Circulating levels of VCAM-1 may predict subsequent clinical cardiovascular events (63) while VCAM-1 may be elevated in T2DM, perhaps as a result of the hypertriglyceridemia and/or low HDL cholesterol (35,64,65). Reducing TG levels with fibrates or fish oil, both of which can be considered PPAR activators (66,67), reportedly decreases soluble adhesion molecule levels (35,67,68). VCAM-1 expression is controlled by multiple pathways, including NF-B and PPAR (69,70). In vitro, VCAM-1 repression has been reported by some PPAR agonists but not others, and at drug concentrations that may have PPAR-independent effects (31–33). Although we found PPAR agonists repressed VCAM-1 in wild-type but not PPAR-deficient ECs, rosiglitazone and 15d-PGJ2 had no VCAM-1 effects (21). Differences among reports of PPAR agonist VCAM-1 effects may involve differences among agents or the cell types under study.

NF-B activation, which is inhibited by IB, increases VCAM-1 expression. PPAR activation induces IB (35). Here we found that pioglitazone increased IB expression in a PPAR-dependent manner in HSVECs in vitro and in liver in vivo; 15 deoxyprostaglandin J2 and troglitazone increase IB expression but independent of PPAR (71–73). The more selective PPAR ligand rosiglitazone did not change IB expression in human monocyte/macrophages (73,74). In contrast, pioglitazone reportedly increased IB levels in peripheral mononuclear cells in human subjects (75). These results are potentially consistent with pioglitazone exerting effects through PPAR as also suggested by studies in which pioglitazone treatment increased expression of PPAR target genes in subcutaneous fat (44). The possibility that these responses derived from pioglitazone activation of PPAR was not discussed (44).

PPAR biology suggests several mechanisms for how agonists for the same PPAR isotype might exert distinct effects. PPARs have a particularly large LBD, even as compared with other nuclear receptors (76). PPAR activation induces a conformational change in the AF2 domain, which allows coactivator recruitment, corepressor release, and formation of the heterodimeric PPAR-RXR complex. These critical determinants of transcriptional responses can vary as a function of different interactions between structurally distinct PPAR agonists and the large PPAR-LBD (76). Interestingly, other in vitro pharmacologic studies also suggest pioglitazone may activate PPAR (36). Recently, ligand-independent mechanisms influencing PPAR-mediated anti-inflammatory effects have been reported, for example through SUMOylation (Small Ubiquitin-related Modifier) (77,78). Differences among PPAR-interacting molecules underlie the concept of selective nuclear receptor modulators, as has been raised for the estrogen receptor and novel PPAR agents in development (79,80). The potential variability among specific PPAR-interacting molecules is apparent in the reports of full agonists, selective partial agonists, inverse agonists, antagonists, as well as pan-PPAR-and dual PPAR agonists (80–83).

Dual PPAR/ agonists offered the putative clinical benefits of combining HDL-raising/TG-lowering via PPAR with improved insulin sensitivity through PPAR (80). No dual PPAR/ agonists have yet been approved for use (84). Muraglitazar and tesaglitazar reached late stage testing before being abandoned due to adverse effects, including increased cardiovascular events (85,86), raising concerns over dual PPAR/ agonists as a drug class. Given the evidence that pioglitazone can be used safely (13), our data that pioglitazone may act, at least in part, through PPAR suggest that selective modulators targeting both PPAR and - may be able to be safely developed. Moreover, our findings suggest pioglitazone's description as a PPAR-specific agonist may need revisiting. Further studies would need to include analysis of pioglitazone's metabolites with biologic activity but unknown PPAR selectivity (87,88). Pioglitazone metabolite production may differ depending on cell types, tissues, species, or genetic variants. Although pioglitazone demonstrates more potent LBD activation of PPAR than PPAR, the PPAR-LBD activation seen is within a range that could influence biologic responses, especially in EC. LBD activation may also underestimate functional PPAR effects in vivo as a result of mechanisms such as preferential generation, stabilization, or transport of a specific drug metabolite.

Pioglitazone could also regulate PPAR target genes indirectly, for example altering PPAR regulatory proteins or inducing the formation of endogenous PPAR agonists. For example, lipoprotein lipase (LPL), a positively regulated PPAR target gene, can generate PPAR ligands through VLDL hydrolysis (22). Increased LPL expression and activity would be associated with lower TG and higher HDL, as occurs with gain of function LPL polymorphisms and after treatment with synthetic PPAR agonists (66,89). Prior work identifies increased LPL-mediated lipolysis as a contributor to pioglitazone's TG-lowering effects (90). In clinical studies, pioglitazone induces LPL expression and also decreases the natural LPL inhibitor apoCIII (44,90), further supporting possible indirect PPAR activation through increased VLDL hydrolysis. Since both LPL and apoCIII are PPAR-regulated target genes, positive feed-forward mechanisms may amplify these effects (22,91).

Independent of a direct or indirect mechanism, PPAR is required in order for VCAM-1 repression and IB induction to occur in vitro and in vivo in mice, which expands potential mechanisms of action for this agent, at least in vitro and in mouse models. This data also underscores the need to fully understand the effects of both existing and emerging PPAR agonists and their biologically active metabolites. Indeed, the complexity of PPAR biology, the number of variables dictating transcriptional and, hence, clinical responses, and the fact that agonist structure can determine biologic response argues that the notion of general PPAR-activating drug classes may be limited.

	Appendix

Top Abstract Methods Results Discussion Appendix References

 
For a supplementary table on the impact of various baseline parameters on sVCAM-1 changes and the sVCAM-1 levels in pioglitazone versus placebo groups after controlling for age, as well as a supplementary figure on Northern blot analysis of PPAR mRNA expression and the quantification of PPAR expression levels by quantitative RT-PCR after concentration-dependent PPAR transfection, please see the online version of this article.

	Acknowledgments

 
The authors thank Ruzena Tupy for excellent editorial assistance.

	Footnotes

 
The clinical trial presented here was supported by Takeda Pharmaceuticals (Dr. Horton, primary investigator). The investigators have received prior funding from GlaxoSmithKline and Takeda Pharmaceuticals, although not specifically for the support of the pre-clinical murine studies included here.

	References

Top Abstract Methods Results Discussion Appendix References

 
1. Glass CK, Witztum JL. Atherosclerosis. The road ahead. Cell 2001;104:503-516.[CrossRef][Web of Science][Medline]

2. Libby P. Current concepts of the pathogenesis of the acute coronary syndromes Circulation 2001;104:365-372.[Free Full Text]

3. Semenkovich CF. Insulin resistance and atherosclerosis J Clin Invest 2006;116:1813-1822.[CrossRef][Web of Science][Medline]

4. Brown JD, Plutzky J. Peroxisome proliferator-activated receptors as transcriptional nodal points and therapeutic targets Circulation 2007;115:518-533.[Abstract/Free Full Text]

5. Lindberg M, Astrup A. The role of glitazones in management of type 2 diabetes. A dream or a nightmare?. Obes Rev 2007;8:381-384.[CrossRef][Web of Science][Medline]

6. Tontonoz P, Hu E, Spiegelman BM. Regulation of adipocyte gene expression and differentiation by peroxisome proliferator activated receptor gamma Curr Opin Genet Dev 1995;5:571-576.[CrossRef][Web of Science][Medline]

7. Willson TM, Cobb JE, Cowan DJ, et al. The structure-activity relationship between peroxisome proliferator-activated receptor gamma agonism and the antihyperglycemic activity of thiazolidinediones J Med Chem 1996;39:665-668.[CrossRef][Web of Science][Medline]

8. Thomson PDR Healthcare. Physicians Desk Reference. 61st edition. Seattle, WA: Thomson PDR Healthcare, 2007.

9. Blaschke F, Caglayan E, Hsueh WA. Peroxisome proliferator-activated receptor gamma agonists: their role as vasoprotective agents in diabetes Endocrinol Metab Clin North Am 2006;35:561-574.[CrossRef][Web of Science][Medline]

10. Yki-Jarvinen H. Thiazolidinediones N Engl J Med 2004;351:1106-1118.[Free Full Text]

11. Goldberg RB, Kendall DM, Deeg MA, et al. A comparison of lipid and glycemic effects of pioglitazone and rosiglitazone in patients with type 2 diabetes and dyslipidemia Diabetes Care 2005;28:1547-1554.[Abstract/Free Full Text]

12. Nissen SE, Wolski K. Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes N Engl J Med 2007;356:2457-2471.[Abstract/Free Full Text]

13. Lincoff AM, Wolski K, Nicholls SJ, Nissen SE. Pioglitazone and risk of cardiovascular events in patients with type 2 diabetes mellitus: a meta-analysis of randomized trials JAMA 2007;298:1180-1188.[Abstract/Free Full Text]

14. Singh S, Loke YK, Furberg CD. Long-term risk of cardiovascular events with rosiglitazone: a meta-analysis JAMA 2007;298:1189-1195.[Abstract/Free Full Text]

15. Diamond GA, Bax L, Kaul S. Uncertain effects of rosiglitazone on the risk for myocardial infarction and cardiovascular death Ann Intern Med 2007;147:578-581.[Abstract/Free Full Text]

16. Krall RL. Cardiovascular safety of rosiglitazone Lancet 2007;369:1995-1996.[CrossRef][Web of Science][Medline]

17. Dormandy JA, Charbonnel B, Eckland DJ, et al. Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive study (PROspective pioglitAzone Clinical Trial In macroVascular Events): a randomised controlled trial Lancet 2005;366:1279-1289.[CrossRef][Web of Science][Medline]

18. Ahmed I, Furlong K, Flood J, Treat VP, Goldstein BJ. Dual PPAR- alpha/gamma agonists: promises and pitfalls in type 2 diabetes Am J Ther 2007;14:49-62.[CrossRef][Medline]

19. Rubenstrunk A, Hanf R, Hum DW, Fruchart JC, Staels B. Safety issues and prospects for future generations of PPAR- modulators Biochim Biophys Acta 2007;177:1065-1081.

20. Galkina E, Ley K. Vascular adhesion molecules in atherosclerosis Arterioscler Thromb Vasc Biol 2007;27:2292-2301.[Abstract/Free Full Text]

21. Marx N, Sukhova GK, Collins T, Libby P, Plutzky J. PPAR-alpha activators inhibit cytokine-induced vascular cell adhesion molecule-1 expression in human endothelial cells Circulation 1999;99:3125-3131.[Abstract/Free Full Text]

22. Ziouzenkova O, Perrey S, Marx N, Bacqueville D, Plutzky J. Peroxisome proliferator-activated receptors Curr Atheroscler Rep 2002;4:59-64.[Medline]

23. Lee SS, Gonzalez FJ. Targeted disruption of the peroxisome proliferator-activated receptor alpha gene, PPAR-alpha Ann N Y Acad Sci 1996;804:524-529.[Web of Science][Medline]

24. Hamdy O, Ledbury S, Mullooly C, et al. Lifestyle modification improves endothelial function in obese subjects with the insulin resistance syndrome Diabetes Care 2003;26:2119-2125.[Abstract/Free Full Text]

25. Grundy SM, Cleeman JI, Merz CN, et al. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines J Am Coll Cardiol 2004;44:720-732.[Abstract/Free Full Text]

26. Tseng KH. Standards of medical care in diabetes—2006 Diabetes Care 2006;29 Suppl 1:S4-S42.

27. Ziouzenkova O, Perrey S, Asatryan L, et al. Lipolysis of triglyceride-rich lipoproteins generates PPAR-ligands: evidence for an antiinflammatory role for lipoprotein lipase Proc Natl Acad Sci U S A 2003;100:2730-2735.[Abstract/Free Full Text]

28. Ahmed W, Orasanu G, Nehra V, et al. High-density lipoprotein hydrolysis by endothelial lipase activates PPAR-alpha: a candidate mechanism for high-density lipoprotein-mediated repression of leukocyte adhesion Circ Res 2006;98:490-498.[Abstract/Free Full Text]

29. Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 2003;26:S5–20.

30. Eckland D, Danhof M. Clinical pharmacokinetics of pioglitazone Exp Clin Endocrinol Diabetes 2000;108 Suppl 2:S234-S242.

31. Jackson SM, Parhami F, Xi XP, et al. Peroxisome proliferator-activated receptor activators target human endothelial cells to inhibit leukocyte-endothelial cell interaction Arterioscler Thromb Vasc Biol 1999;19:2094-2104.[Abstract/Free Full Text]

32. Li AC, Brown KK, Silvestre MJ, Willson TM, Palinski W, Glass CK. Peroxisome proliferator-activated receptor gamma ligands inhibit development of atherosclerosis in LDL receptor-deficient mice J Clin Invest 2000;106:523-531.[Web of Science][Medline]

33. Rival Y, Beneteau N, Taillandier T, et al. PPAR-alpha and PPAR-delta activators inhibit cytokine-induced nuclear translocation of NF-kappaB and expression of VCAM-1 in EAhy926 endothelial cells Eur J Pharmacol 2002;435:143-151.[CrossRef][Web of Science][Medline]

34. Dreyer C, Krey G, Keller H, Givel F, Helftenbein G, Wahli W. Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors Cell 1992;68:879-887.[CrossRef][Web of Science][Medline]

35. Delerive P, Gervois P, Fruchart JC, Staels B. Induction of IkappaBalpha expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor-alpha activators J Biol Chem 2000;275:36703-36707.[Abstract/Free Full Text]

36. Sakamoto J, Kimura H, Moriyama S, et al. Activation of human peroxisome proliferator-activated receptor (PPAR-) subtypes by pioglitazone Biochem Biophys Res Commun 2000;278:704-711.[CrossRef][Web of Science][Medline]

37. Ryan KE, McCance DR, Powell L, McMahon R, Trimble ER. Fenofibrate and pioglitazone improve endothelial function and reduce arterial stiffness in obese glucose tolerant men Atherosclerosis 2007;194:e123-e130.[CrossRef][Medline]

38. Takase H, Nakazawa A, Yamashita S, et al. Pioglitazone produces rapid and persistent reduction of vascular inflammation in patients with hypertension and type 2 diabetes mellitus who are receiving angiotensin II receptor blockers Metabolism 2007;56:559-564.[CrossRef][Web of Science][Medline]

39. Gao J, Ann Garulacan L, Storm SM, et al. Identification of in vitro protein biomarkers of idiosyncratic liver toxicity Toxicol In Vitro 2004;18:533-541.[CrossRef][Web of Science][Medline]

40. Bottoni P, Giardina B, Martorana GE, et al. A two-dimensional electrophoresis preliminary approach to human hepatocarcinoma differentiation induced by PPAR–agonists J Cell Mol Med 2005;9:462-467.[CrossRef][Web of Science][Medline]

41. Guo L, Zhang L, Sun Y, et al. Differences in hepatotoxicity and gene expression profiles by anti-diabetic PPAR- gamma agonists on rat primary hepatocytes and human HepG2 cells Mol Divers 2006;10:349-360.[CrossRef][Web of Science][Medline]

42. Johnson MD, Campbell LK, Campbell RK. Troglitazone: review and assessment of its role in the treatment of patients with impaired glucose tolerance and diabetes mellitus Ann Pharmacother 1998;32:337-348.[Abstract]

43. Chiquette E, Ramirez G, Defronzo R. A meta-analysis comparing the effect of thiazolidinediones on cardiovascular risk factors Arch Intern Med 2004;164:2097-2104.[Abstract/Free Full Text]

44. Bogacka I, Xie H, Bray GA, Smith SR. The effect of pioglitazone on peroxisome proliferator-activated receptor-gamma target genes related to lipid storage in vivo Diabetes Care 2004;27:1660-1667.[Abstract/Free Full Text]

45. Plutzky J. Peroxisome proliferator-activated receptors in endothelial cell biology Curr Opin Lipidol 2001;12:511-518.[CrossRef][Web of Science][Medline]

46. Haffner SM, Greenberg AS, Weston WM, Chen H, Williams K, Freed MI. Effect of rosiglitazone treatment on nontraditional markers of cardiovascular disease in patients with type 2 diabetes mellitus Circulation 2002;106:679-684.[Abstract/Free Full Text]

47. Sidhu JS, Cowan D, Kaski JC. The effects of rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, on markers of endothelial cell activation, C-reactive protein, and fibrinogen levels in non-diabetic coronary artery disease patients J Am Coll Cardiol 2003;42:1757-1763.[Abstract/Free Full Text]

48. Plutzky J. Peroxisome proliferator-activated receptors as therapeutic targets in inflammation J Am Coll Cardiol 2003;42:1764-1766.[Free Full Text]

49. Yoshimoto T, Naruse M, Shizume H, et al. Vasculo-protective effects of insulin sensitizing agent pioglitazone in neointimal thickening and hypertensive vascular hypertrophy Atherosclerosis 1999;145:333-340.[CrossRef][Web of Science][Medline]

50. Koshiyama H, Shimono D, Kuwamura N, Minamikawa J, Nakamura Y. Rapid communication: inhibitory effect of pioglitazone on carotid arterial wall thickness in type 2 diabetes J Clin Endocrinol Metab 2001;86:3452-3456.[Abstract/Free Full Text]

51. Sidhu JS, Kaposzta Z, Markus HS, Kaski JC. Effect of rosiglitazone on common carotid intima-media thickness progression in coronary artery disease patients without diabetes mellitus Arterioscler Thromb Vasc Biol 2004;24:930-934.[Abstract/Free Full Text]

52. Mazzone T, Meyer PM, Feinstein SB, et al. Effect of pioglitazone compared with glimepiride on carotid intima-media thickness in type 2 diabetes: a randomized trial JAMA 2006;296:2572-2581.[Abstract/Free Full Text]

53. Hedblad B, Zambanini A, Nilsson P, Janzon L, Berglund G. Rosiglitazone and carotid IMT progression rate in a mixed cohort of patients with type 2 diabetes and the insulin resistance syndrome: main results from the Rosiglitazone Atherosclerosis study J Intern Med 2007;261:293-305.[CrossRef][Web of Science][Medline]

54. Dandona P, Aljada A, Chaudhuri A. Vascular reactivity and thiazolidinediones Am J Med 2003;115:81S-86S.[Web of Science]

55. Lago RM, Singh PP, Nesto RW. Congestive heart failure and cardiovascular death in patients with prediabetes and type 2 diabetes given thiazolidinediones: a meta-analysis of randomised clinical trials Lancet 2007;370:1129-1136.[CrossRef][Web of Science][Medline]

56. Nissen SE, Nicholls SJ, Wolski K, et al. Comparison of pioglitazone vs glimepiride on progression of coronary atherosclerosis in patients with type 2 diabetes: the PERISCOPE randomized controlled trial JAMA 2008;299:1561-1573.[Abstract/Free Full Text]

57. Dierkes J, Westphal S, Luley C. Serum homocysteine increases after therapy with fenofibrate or bezafibrate Lancet 1999;554:219-220.

58. Lipscombe J, Lewis GF, Cattran D, Bargman JM. Deterioration in renal function associated with fibrate therapy Clin Nephrol 2001;55:39-44.[Web of Science][Medline]

59. Luc G, Jacob N, Bouly M, Fruchart JC, Staels B, Giral P. Fenofibrate increases homocystinemia through a PPAR-alpha-mediated mechanism J Cardiovasc Pharmacol 2004;43:452-453.[CrossRef][Web of Science][Medline]

60. Davidson MH, Armani A, McKenney JM, Jacobson TA. Safety considerations with fibrate therapy Am J Cardiol 2007;99:3C-18C.[Web of Science][Medline]

61. Rubins HB, Robins SJ, Collins D, et al. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. N Engl J Med 1999;341:410-418.[Abstract/Free Full Text]

62. Keech A, Simes RJ, Barter P, et al. Effects of long-term fenofibrate therapy on cardiovascular events in 9795 people with type 2 diabetes mellitus (the FIELD study): randomised controlled trial Lancet 2005;366:1849-1861.[CrossRef][Web of Science][Medline]

63. Mulvihill NT, Foley JB, Crean P, Walsh M. Prediction of cardiovascular risk using soluble cell adhesion molecules Eur Heart J 2002;23:1569-1574.[Free Full Text]

64. Abe Y, El-Masri B, Kimball KT, et al. Soluble cell adhesion molecules in hypertriglyceridemia and potential significance on monocyte adhesion Arterioscler Thromb Vasc Biol 1998;18:723-731.[Abstract/Free Full Text]

65. Calabresi L, Gomaraschi M, Villa B, Omoboni L, Dmitrieff C, Franceschini G. Elevated soluble cellular adhesion molecules in subjects with low HDL-cholesterol Arterioscler Thromb Vasc Biol 2002;22:656-661.[Abstract/Free Full Text]

66. Pineda Torra I, Gervois P, Staels B. Peroxisome proliferator-activated receptor alpha in metabolic disease, inflammation, atherosclerosis and aging Curr Opin Lipidol 1999;10:151-159.[CrossRef][Web of Science][Medline]

67. Sethi S, Ziouzenkova O, Ni H, Wagner DD, Plutzky J, Mayadas TN. Oxidized omega-3 fatty acids in fish oil inhibit leukocyte-endothelial interactions through activation of PPAR-alpha Blood 2002;100:1340-1346.[Abstract/Free Full Text]

68. Okapcova J, Gabor D. The levels of soluble adhesion molecules in diabetic and nondiabetic patients with combined hyperlipoproteinemia and the effect of ciprofibrate therapy Angiology 2004;55:629-639.[CrossRef][Medline]

69. Xu X, Otsuki M, Saito H, et al. PPAR-alpha and GR differentially down-regulate the expression of nuclear factor-kappaB-responsive genes in vascular endothelial cells Endocrinology 2001;142:3332-3339.[Abstract/Free Full Text]

70. Blankenberg S, Barbaux S, Tiret L. Adhesion molecules and atherosclerosis Atherosclerosis 2003;170:191-203.[CrossRef][Web of Science][Medline]

71. Rossi A, Kapahi P, Natoli G, et al. Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase Nature 2000;403:103-108.[CrossRef][Medline]

72. Ward C, Dransfield I, Murray J, Farrow SN, Haslett C, Rossi AG. Prostaglandin D2 and its metabolites induce caspase-dependent granulocyte apoptosis that is mediated via inhibition of I kappa B alpha degradation using a peroxisome proliferator-activated receptor-gamma-independent mechanism J Immunol 2002;168:6232-6243.[Abstract/Free Full Text]

73. Guyton K, Bond R, Reilly C, Gilkeson G, Halushka P, Cook J. Differential effects of 15-deoxy-delta(12,14)-prostaglandin J2 and a peroxisome proliferator-activated receptor gamma agonist on macrophage activation J Leukoc Biol 2001;69:631-638.[Abstract/Free Full Text]

74. Mohanty P, Aljada A, Ghanim H, et al. Evidence for a potent antiinflammatory effect of rosiglitazone J Clin Endocrinol Metab 2004;89:2728-2735.[Abstract/Free Full Text]

75. Klotz L, Schmidt M, Giese T, et al. Proinflammatory stimulation and pioglitazone treatment regulate peroxisome proliferator-activated receptor gamma levels in peripheral blood mononuclear cells from healthy controls and multiple sclerosis patients J Immunol 2005;175:4948-4955.[Abstract/Free Full Text]

76. Willson TM, Brown PJ, Sternbach DD, Henke BR. The PPAR-s: from orphan receptors to drug discovery J Med Chem 2000;43:527-550.[CrossRef][Web of Science][Medline]

77. Pascual G, Fong AL, Ogawa S, et al. A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR–gamma Nature 2005;437:759-763.[CrossRef][Medline]

78. Ghisletti S, Huang W, Ogawa S, et al. Parallel SUMOylation-dependent pathways mediate gene- and signal-specific transrepression by LXRs and PPAR-gamma Mol Cell 2007;25:57-70.[CrossRef][Web of Science][Medline]

79. Dutertre M, Smith CL. Molecular mechanisms of selective estrogen receptor modulator (SERM) action J Pharmacol Exp Ther 2000;295:431-437.[Abstract/Free Full Text]

80. Fievet C, Fruchart JC, Staels B. PPAR-alpha and PPAR-gamma dual agonists for the treatment of type 2 diabetes and the metabolic syndrome Curr Opin Pharmacol 2006;6:606-614.[CrossRef][Web of Science][Medline]

81. Knouff C, Auwerx J. Peroxisome proliferator-activated receptor-gamma calls for activation in moderation: lessons from genetics and pharmacology Endocr Rev 2004;25:899-918.[Abstract/Free Full Text]

82. Seimandi M, Lemaire G, Pillon A, et al. Differential responses of PPAR-alpha, PPAR-delta, and PPAR-gamma reporter cell lines to selective PPAR-synthetic ligands Anal Biochem 2005;344:8-15.[CrossRef][Web of Science][Medline]

83. Hamuro Y, Coales SJ, Morrow JA, et al. Hydrogen/deuterium-exchange (H/D-Ex) of PPAR-gamma LBD in the presence of various modulators Protein Sci 2006;15:1883-1892.[CrossRef][Web of Science][Medline]

84. Henke BR. Peroxisome proliferator-activated receptor alpha/gamma dual agonists for the treatment of type 2 diabetes J Med Chem 2004;47:4118-4127.[CrossRef][Web of Science][Medline]

85. Nissen SE, Wolski K, Topol EJ. Effect of muraglitazar on death and major adverse cardiovascular events in patients with type 2 diabetes mellitus JAMA 2005;294:2581-2586.[Abstract/Free Full Text]

86. Hellmold H, Zhang H, Andersson U, et al. Tesaglitazar, a PPAR-alpha/gamma agonist, induces interstitial mesenchymal cell DNA synthesis and fibrosarcomas in subcutaneous tissues in rats Toxicol Sci 2007;98:63-74.[Abstract/Free Full Text]

87. Lin ZJ, Ji W, Desai-Krieger D, Shum L. Simultaneous determination of pioglitazone and its two active metabolites in human plasma by LC-MS/MS J Pharm Biomed Anal 2003;33:101-108.[CrossRef][Web of Science][Medline]

88. Shen Z, Reed JR, Creighton M, et al. Identification of novel metabolites of pioglitazone in rat and dog Xenobiotica 2003;33:499-509.[CrossRef][Web of Science][Medline]

89. Wang XL, McCredie RM, Wilcken DE. Common DNA polymorphisms at the lipoprotein lipase gene. Association with severity of coronary artery disease and diabetes. Circulation 1996;96:1339-1345.[Web of Science]

90. Nagashima K, Lopez C, Donovan D, et al. Effects of the PPAR-gamma agonist pioglitazone on lipoprotein metabolism in patients with type 2 diabetes mellitus J Clin Invest 2005;115:1323-1332.[CrossRef][Web of Science][Medline]

91. Staels B, Vu-Dac N, Kosykh VA, et al. Fibrates downregulate apolipoprotein C-III expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the hypolipidemic action of fibrates. J Clin Invest 1995;95:705-712.[Web of Science][Medline]

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