PPARalpha activators inhibit cytokine-induced vascular cell adhesion molecule-1 expression in human endothelial cells

Abstract

Background: Adhesion molecule expression on the endothelial cell (EC) surface is critical for leukocyte recruitment to atherosclerotic lesions. Better understanding of transcriptional regulation of adhesion molecules in ECs may provide important insight into plaque formation. Peroxisome proliferator-activated receptor-alpha (PPARalpha), a member of the nuclear receptor family, regulates gene expression in response to certain fatty acids and fibric acid derivatives. The present study investigated PPARalpha expression in human ECs and their regulation of vascular cell adhesion molecule-1 (VCAM-1).

Methods and results: Immunohistochemistry revealed that human carotid artery ECs express PPARalpha. Pretreatment of cultured human ECs with the PPARalpha activators fenofibrate or WY14643 inhibited TNF-alpha-induced VCAM-1 in a time- and concentration-dependent manner, an effect not seen with PPARgamma activators. Both PPARalpha activators decreased cytokine-induced VCAM-1 mRNA expression without altering its mRNA half-life. Transient transfection of deletional VCAM-1 promoter constructs and electrophoretic mobility shift assays suggest that fenofibrate inhibits VCAM-1 transcription in part by inhibiting NF-kappaB. Finally, PPARalpha activators significantly reduced adhesion of U937 cells to cultured human ECs.

Conclusions: Human ECs express PPARalpha, a potentially important regulator of atherogenesis through its transcriptional control of VCAM-1 gene expression. Such findings also have implications regarding the clinical use of lipid-lowering agents, like fibric acids, which can activate PPARalpha.

Figure 1

ECs express PPARα in vivo and in vitro. A, PPARα expression in nuclei of ECs of human carotid arteries (positive nuclei stained blue, arrowheads). B, Parallel sections stained with goat IgG show no signal, which suggests that staining for PPARα is specific. C, Immunostaining of parallel sections with CD31 identifies endothelial cell layer at luminal surface of artery (stained red). Magnification ×400 in all. Analysis of 6 separate carotid sections revealed similar results. D, Western blot analysis of cultured human saphenous vein ECs reveals PPARα protein expression in nuclear extracts (Nucl). Identity of detected band was confirmed by comigration with a band from fibroblasts transfected with a PPARα expression construct (Co). PPARα is not seen in cytosolic fraction of ECs (Cyto). Three independent experiments showed similar results.

Figure 2

PPARα but not PPARγ activators inhibit cytokine-induced cell surface expression of VCAM-1 in human ECs. A and B, Cells were pretreated with PPARα [100 µmol/L fenofibrate (feno), 250 µmol/L WY14643 (WY)] or PPARγ [10 µmol/L troglitazone (trogl), 10 µmol/L 15d-PGJ2 (PGJ2), 10 µmol/L BRL49653 (BRL)] activators for 24 hours and then stimulated with TNF-α 10 µg/L for 8 hours before cell surface EIAs were performed for VCAM-1 (A), ICAM-1, and E-selectin (B). Results are expressed as percent of TNF-α–stimulated cells (% control). Bars represent mean±SEM (VCAM-1, n=8; ICAM-1/E-selectin, n=4); *P<0.05, **P<0.01 vs control. C and D, PPARα activators inhibit surface expression of VCAM-1 in human ECs in a time- and concentration-dependent manner. Cells were pretreated with fenofibrate 100 µmol/L for duration shown before stimulation with TNF-α 10 µg/L for 8 hours (C). ECs were pretreated with fenofibrate at concentrations shown for 24 hours, before stimulation with TNF-α 10 µg/L (D, solid bars), or IL-1α 10 µg/L (D, open bars). Results are expressed as percent of cytokine-stimulated cells (% control) as determined by cell surface EIA. Circles/bars represent mean±SEM (n=3); *P<0.05, **P<0.01 vs control.

Figure 3

PPARα activators inhibit adhesion of U937 monocytoid cells to human ECs. A, Fluorescein-labeled U937 cells were added to TNF-α 10 µg/L–stimulated human EC monolayers with or without fenofibrate pretreatment. Fluorescence microscopy shows adherent U937 cells (green) on ECs. B, Quantification of U937 adherence on EC monolayers after EC pretreatment with fenofibrate (feno) 100 µmol/L or WY14643 (WY) 250 µmol/L as determined by fluorescence assay. Results are expressed as cells/cm². Bars represent mean±SEM (n=3); **P<0.01 vs TNF-α stimulated cells.

Figure 4

PPARα activators inhibit VCAM-1 mRNA expression but not mRNA half-life in human ECs. A, Northern blot analysis of ECs pretreated with fenofibrate or WY14643 at concentrations shown for 24 hours before stimulation with TNF-α 10 µg/L for 3 hours (top). Ethidium bromide staining (bottom) demonstrates equal loading of intact RNA. Three independent experiments yielded similar results. B, Densitometry analysis of TNF-α–induced VCAM-1 mRNA levels in absence or presence of fenofibrate 100 µmol/L as measured by Northern blot analysis of actinomycin D-treated ECs. Actinomycin D and fenofibrate were added to ECs 3 hours after TNF-α stimulation (0 hours); cells were harvested at times indicated. Amount of mRNA at each time point was compared with mRNA levels after 3 hours of TNF-α stimulation at time 0 hours (ordinate labeled as relative mRNA level). Results are shown as mean6SEM of 3 independent experiments.

Figure 5

Fenofibrate inhibits TNF-α–induced activation of human VCAM-1 promoter and activation of NF-κB. A, Deletional series of VCAM-1 promoter constructs used in transfection experiments. B, Bovine ECs were cotransfected with VCAM-1 promoter deletion constructs indicated and a β-galactosidase expression construct (pCMV.β-GAL). Transfected cells were stimulated with agents indicated for 36 hours, and assays were performed for CAT- and β-galactosidase activity. Results for each construct were normalized to b-galactosidase activity and expressed as multiples of induction compared with unstimulated cells. Bars represent mean±SEM (n=3); *P<0.05 vs TNF-α– stimulated cells. C, EMSA of human ECs pretreated with fenofibrate 100 µmol/L for 24 hours before TNF-α stimulation for 2 hours. Specificity was determined by addition of 40 ng unlabeled NF-κB oligonucleotide (cold probe). Supershift analysis was performed with anti-p50 and anti-p65 antibodies. As a control, nuclear extracts were incubated with nonspecific IgG. Similar results were seen in 3 independent experiments.