Abstract
Recent evidence shows that peroxisome proliferator-activated receptor gamma (PPARgamma) ligands induce the antiangiogenic effect in endothelial cells and tumors. In the present study, we elucidated the involvement of maxi-K channel activation in the antiangiogenic effect of rosiglitazone, a well known PPARgamma ligand in human umbilical vein endothelial cells. We found that the antiangiogenic effects of rosiglitazone were reversed by either bisphenol A diaglycidyl ether, a PPARgamma antagonist, or iberiotoxin, a maxi-K channel blocker. Knockdown of maxi-K channel expression also reversed the antiangiogenic effects. Iberiotoxin reversed the rosiglitazone-induced hyperpolarization while having no effect on the endogenous PPARgamma activation, suggesting that rosiglitazone activates maxi-K channel via PPARgamma. In the rosiglitazone-induced antiangiogenic process, endothelial nitric-oxide synthase-Ser1179 phosphorylation and NO production were significantly elevated, and treatment with the NOS inhibitor N(G)-monomethyl-L-arginine acetate abolished the antiangiogenic and apoptotic effects of rosiglitazone, indicating NO as a key mediator of the rosiglitazone actions. In conclusion, rosiglitazone significantly inhibited VEGF165-induced angiogenesis by a proapoptotic mechanism via PPARgamma-mediated NO production, followed by maxi-K channel opening.
Highlights
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily and bind to specific DNA sequences to induce transcriptional activation of specific genes [6]
Rosiglitazone (10 M) significantly reduced the formation of VEGF165-stimulated neomicrovessels and hemoglobin contents (57% inhibition; VEGF165 only, 6.12 Ϯ 0.60 g/dl; rosiglitazone ϩ VEGF165, 2.44 Ϯ 0.47 g/dl), which was completely antagonized by either bisphenol A diaglycidyl ether (BADGE) or iberiotoxin (Fig. 2F). These results suggest that rosiglitazone elicited a strong in vivo antiangiogenic activity via PPAR␥-dependent maxi-K channel opening
PPAR␥ has been recognized as a potential therapeutic target for the treatment of pathologic neovascularization [13, 25], since various PPAR␥ ligands inhibited growth and/or migration of vascular endothelial cells, smooth muscle cells, monocytes, and certain tumor cells [27, 28]
Summary
Cell Cultures—HUVEC (ATCC CRL-1730; endothelial cell line derived from vein of human umbilical cord) were cultured in Kaighn’s F12K medium supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mg/ml heparin sodium, 0.03–0.05 mg/ml endothelial cell growth supplement, and 1% antibiotics (100 units/ml penicillin and 100 g/ml streptomycin). Membrane Potential Measurement Using Voltage-sensitive Fluorescent Dye—Cells (1.5 ϫ 104 cells/well) were incubated with 200 nM DiBAC in Dulbecco’s phosphate-buffered saline for 30 min at 37 °C and exposed to BADGE (20 M), iberiotoxin (0.3 M), or NG-monomethylL-arginine acetate (L-NMMA, 10 M) for 1 h and stimulated by rosiglitazone or NS-1619 (10 M) for 3 h, followed by VEGF165 (10 ng/ml) for 1 h. Measurement of Cytosolic Ca2ϩ and NO Concentrations—Cells transfected with either control RNAi or Maxi-K channel StealthTM RNAi were seeded at 5 ϫ 104 cells/well in 12-well tissue culture plates and incubated with Kaighn’s F12K medium containing 1% fetal bovine serum plus BADGE or iberiotoxin for 1 h and exposed to rosiglitazone or NS-1619 for 3 h. Student’s t test was used for analyzing values between vehicle groups and compoundtreated groups. p Ͻ 0.05 was considered to be statistically significant
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