Fig. 4. PPARδ was required for PGD₂- and 15-d-PGJ₂-dependent VSMC phenotypic changes.

a P4-stage VSMCs were stimulated with the indicated PGD₂ metabolites, and the expression levels of SMA and SM22α were assessed by western blot analysis. b The promoter activity of PPRE was verified in the presence of the indicated stimuli (n = 3). c P4-stage VSMCs were pretreated with a PPARα, PPARδ, or PPARγ antagonist (GW6471, GSK3787, or GW9662) and then stimulated with 15-d-PGJ₂. The expression levels of SMA and SM22α were assessed by western blot analysis. d Contractile VSMCs were infected with a retrovirus carrying either vector or the PPARδ construct, and the promoter activity of PPRE was verified in the presence of the indicated stimuli (n = 3). GW501516 (10 μM) was used as a positive control for the PPARδ agonist. e FLAG-tagged PPARδ was expressed in P4-stage VSMCs, and the expression levels of SMA and SM22α were assessed after stimulation with 15-d-PGJ₂. f PPARδ was silenced in P4-stage VSMCs, and the expression of SMA and SM22α was assessed after cells were stimulated with 15-d-PGJ₂. g The proliferation of P0- or P4-stage VSMCs was measured (n = 10). h Vector or PPARδ was expressed in P4-stage VSMCs, and proliferation was measured (n = 5). i Aortic tissues were isolated, the endothelial and adventitial layers were discarded, and the aortic tissue fragments were placed on collagen-coated plates and then stimulated with 15-d-PGJ₂ for 7 days. Proliferating VSMCs are indicated by dashed lines. The results are presented as the means ± SEM. One-way ANOVA and Tukey’s multiple comparison test were used to determine the P values. The asterisks indicate statistical significance (P < 0.05).