(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).

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.

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.

(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.

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).

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.