J Lipid Atheroscler.  2012 Jun;1(1):11-20. 10.12997/jla.2012.1.1.11.

The Peroxisome Proliferator-Activated Receptor delta Agonist, GW501516, Inhibits Angiogenesis through Dephosphorylation of Endothelial Nitric Oxide Synthase

• Kim JB
• Lee SH
• Ahn J
• Kim J

• KMID: 2198371
• DOI: http://doi.org/10.12997/jla.2012.1.1.11

Abstract

OBJECTIVE
Peroxisome proliferator-activated receptor delta (PPAR-delta) is an ubiquitously expressed nuclear receptor that has been implicated in adipose tissue formation, brain development, and atherosclerosis. Despite mouse studies demonstrating that PPAR-delta activation has favorable anti-atherogenic properties by improving systemic lipid profiles, the relationship between PPAR-delta agonist and angiogenesis is unknown. We hypothesized that PPAR-delta ligands modulate the angiogenesis.
METHODS
To test this hypothesis we treated primary cultures of bovine aortic endothelial cells with PPAR-delta specific ligand, GW501516 (50-800 nM) for 6 h.
RESULTS
GW501516 dose-dependently decreased nitric oxide production without alteration in endothelial nitric oxide synthase (eNOS) expression. Analysis with phospho-specific antibodies against eNOS demonstrated that GW501516 significantly decreased the phosphorylation of eNOS at Serine1179 (eNOS-Ser1179). Concurrently, GW501516 also decreased the Akt phosphorylation. GW501516 did not affect endothelial cell proliferation or induce apoptosis. However, GW501516 inhibited endothelial cell migration, and tube formation in a high nanomolar concentration. The inhibition of endothelial cell tube formation by GW501516 was prevented by addition of the nitric oxide donor, DETA NONOate (5 microM). GW501516 was also found to inhibit angiogenesis in vivo in the chicken chorioallantoic membrane assay.
CONCLUSION
These results provide that high nanomolar range of GW501516 inhibits angiogenesis by a mechanism involving dephosphorylation of eNOS-Ser1179.

Keyword

Aortic endothelial cells; GW501516; Nitric oxide; Angiogenesis

MeSH Terms

Adipose Tissue
Animals
Antibodies, Phospho-Specific
Apoptosis
Atherosclerosis
Brain
Chickens
Chorioallantoic Membrane
DEET
Endothelial Cells
Humans
Ligands
Mice
Nitric Oxide
Nitric Oxide Synthase Type III
Nitroso Compounds
Peroxisomes
Phosphorylation
PPAR delta
Thiazoles
Tissue Donors
Antibodies, Phospho-Specific
DEET
Ligands
Nitric Oxide
Nitric Oxide Synthase Type III
Nitroso Compounds
PPAR delta
Thiazoles

Figure

• 

  Fig. 1 Effect of GW501516 according to dose on NO production in BAECs. (A) Dose-dependent decrease of NO production in BAECs without alteration of eNOS. (B) GW501516 inhibited phosphorylation of eNOS-Ser1179 (C) GW501516 decreased phosphorylation of p-Akt-Ser473. Data are means±SD of triplicate experiments. *P<0.05, **P<0.01 by ANOVA.

• 

  Fig. 2 Effect of GW501516 on cell viability in BAECs. Data are means±SD of triplicate experiments.

• 

  Fig. 3 Effect of GW501516 compared with DMSO on the migration of BAECs. BAECs wounded with a razor were treated with/without 800 nM GW501516 for 16 h and the migrated cells were counted. Migration was quantified by counting the number of cells that moved beyond the injury line. Data are means±SD of triplicate experiments. **P<0.01 by Student's paired t-test.

• 

  Fig. 4 Influence of GW501516 treatment on tube formation of BAECs. The effect of GW501516 on the morphological changes of BAECs on the surface of Matrigel was investigated. BAECs were grown on 48-well plates pre-coated with Matrigel and GW501516 for 6 h after seeding. The endothelial morphological changes were captured through an inverted microscope (40×) and photographed. Data are representative results from three separate experiments.

• 

  Fig. 5 In situ inhibition of angiogenesis in the chicken CAM. Fat emulsion (10%) was injected into the CAM to make the vascular network clear. Data are representative results from three separate experiments.

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