Korean J Physiol Pharmacol.  2011 Apr;15(2):83-88. 10.4196/kjpp.2011.15.2.83.

HO-1 Induced by Cilostazol Protects Against TNF-alpha-associated Cytotoxicity via a PPAR-gamma-dependent Pathway in Human Endothelial Cells

• Park SY
• Bae JU
• Hong KW
• Kim CD

• KMID: 2285411
• DOI: http://doi.org/10.4196/kjpp.2011.15.2.83

Abstract

A large body of evidence has indicated that induction of endogenous antioxidative proteins seems to be a reasonable strategy for delaying the progression of cell injury. In our previous study, cilostazol was found to increase the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) in synovial cells. Thus, the present study was undertaken to examine whether cilostazol is able to counteract tumor necrosis factor-alpha (TNF-alpha)-induced cell death in endothelial cells via the induction of HO-1 expression. We exposed human umbilical vein endothelial cells (HUVECs) to TNF-alpha (50 ng/ml), with or without cilostazol (10 microM). Pretreatment with cilostazol markedly reduced TNF-alpha-induced viability loss in the HUVECs, which was reversed by zinc protoporphyrine IX (ZnPP), an inhibitor of HO-1. Moreover, cilostazol increased HO-1 protein and mRNA expression. Cilostazol-induced HO-1 induction was markedly attenuated not only by ZnPP but also by copper-protoporphyrin IX (CuPP). In an assay measuring peroxisome proliferator-activated receptor-gamma (PPAR-gamma) transcription activity, cilostazol directly increased PPAR-gamma transcriptional activity which was completely abolished by HO-1 inhibitor. Furthermore, increased PPAR-gamma activity by cilostazol and rosiglitazone was completely abolished in cells transfected with HO-1 siRNA. Taken together, these results indicate that cilostazol up-regulates HO-1 and protects cells against TNF-alpha-induced endothelial cytotoxicity via a PPAR-gamma-dependent pathway.

Keyword

Cilosatzol; PPAR-gamma; HO-1; Endothelial cells

MeSH Terms

Cell Death
Endothelial Cells
Heme Oxygenase-1
Human Umbilical Vein Endothelial Cells
Humans
Peroxisomes
Proteins
RNA, Messenger
RNA, Small Interfering
Tetrazoles
Thiazolidinediones
Tumor Necrosis Factor-alpha
Zinc
Heme Oxygenase-1
Proteins
RNA, Messenger
RNA, Small Interfering
Tetrazoles
Thiazolidinediones
Tumor Necrosis Factor-alpha
Zinc

Figure

• 

  Fig. 1. Dose-dependent cytotoxic effects of TNF-α (1∼100 ng/ml) on HUVECs (A) and suppression of these effects by cilostazol (B). Cells were pretreated with cilostazol (1∼100 μM) for 4 h, and then stimulated with TNF-α (50 ng/mL) for 18 h. Results are expressed as the mean±S.E.M. of four experiments. ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001 vs. none, ##p<0.01 vs. TNF-α alone.

• 

  Fig. 2. Effect of an HO-1 inhibitor on the protective effects of cilostazol against TNF-α-mediated cytotoxicity in HUVECs. Cells were pretreated with cilostazol in the presence or absence of an HO-1 inhibitor (ZnPP, 1 μM) and then stimulated with TNF-α (50 ng/ml) for 18 h. Results are expressed as the mean±S.E.M. of four experiments. ∗∗p<0.01 vs. none, #p<0.05 vs. TNF-α alone, ††p<0.01 vs. TNF-α + cilostazol.

• 

  Fig. 3. Cilostazol induces HO-1 expression. HUVECs were stimulated with different concentrations of cilostazol for 12 h. The expression of HO-1 mRNA and protein was determined using reverse transcription PCR and Western blotting, respectively. GAPDH and β-actin were used as loading controls. Results are expressed as the mean±S.E.M. of four experiments. ∗p<0.05, ∗∗p<0.01 vs. vehicle.

• 

  Fig. 4. Effect of an HO-1 inhibitor on cilostazol-induced HO-1 expression. HUVECs were pretreated with an HO-1 inhibitor (ZnPP), and then stimulated with cilostazol (10 μM) for 12 h. The expression of HO-1 mRNA and protein was determined using reverse transcription PCR and Western blotting, respectively. GAPDH and μ-actin were used as loading controls. Results are expressed as the mean±S.E.M. of three or four experiments. ∗p<0.05; ∗∗p<0.01, ∗∗∗p <0.001 vs. vehicle, #p<0.05; ###p <0.001 vs. cilostazol alone.

• 

  Fig. 5. HO-1 mediates the effect of cliostazol and rosiglitazone on PPAR-γ transcriptional activity. (A) HUVECs were transiently transfected with PPRE-pGL3, a renilla luciferase control reporter vector, and then treated either with cilostazol (10 μM) or rosiglitazone (10 μM) for 24 h. PPAR-γ transcriptional activity was determined using a luciferase activity assay. (B.) HUVECs were transfected with siRNA specific for HO-1 then treated with cilostazol (10 μM) or rosiglitazone (10 μM) for 24 h. ∗∗p<0.01, ∗∗∗p<0.001 vs. no treatment, ###p<0.001 vs. cilostazol, †††p<0.001 vs. rosiglitazone.

Reference

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