Objective— We recently reported that the peroxisome proliferator-activated receptor γ (PPARγ) ligands 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) and ciglitazone increased cultured endothelial cell nitric oxide (NO) release without increasing the expression of endothelial nitric oxide synthase (eNOS). The current study was designed to characterize further the molecular mechanisms underlying PPARγ-ligand–stimulated increases in endothelial cell NO production.

Methods and Results— Treating human umbilical vein endothelial cells (HUVEC) with PPARγ ligands (10 μmol/L 15d-PGJ₂, ciglitazone, or rosiglitazone) for 24 hours increased NOS activity and NO release. In selected studies, HUVEC were treated with PPARγ ligands and with the PPARγ antagonist GW9662 (2 μmol/L), which fully inhibited stimulation of a luciferase reporter gene, or with small interfering RNA to PPARγ, which reduced HUVEC PPARγ expression. Treatment with either small interfering RNA to PPARγ or GW9662 inhibited 15d-PGJ₂-, ciglitazone-, and rosiglitazone-induced increases in endothelial cell NO release. Rosiglitazone and 15d-PGJ₂, but not ciglitazone, increased heat shock protein 90-eNOS interaction and eNOS ser¹¹⁷⁷ phosphorylation. The heat shock protein 90 inhibitor geldanamycin attenuated 15d-PGJ₂- and rosiglitazone-stimulated NOS activity and NO production.

Conclusions— These findings further clarify mechanisms involved in PPARγ-stimulated endothelial cell NO release and emphasize that individual ligands exert their effects through distinct PPARγ-dependent mechanisms.