PPARdelta-mediated antiinflammatory mechanisms inhibit angiotensin II-accelerated atherosclerosis

Abstract

Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor delta (PPARdelta) has been shown to improve insulin resistance, adiposity, and plasma HDL levels. However, its antiatherogenic role remains controversial. Here we report atheroprotective effects of PPARdelta activation in a model of angiotensin II (AngII)-accelerated atherosclerosis, characterized by increased vascular inflammation related to repression of an antiinflammatory corepressor, B cell lymphoma-6 (Bcl-6), and the regulators of G protein-coupled signaling (RGS) proteins RGS4 and RGS5. In this model, administration of the PPARdelta agonist GW0742 (1 or 10 mg/kg) substantially attenuated AngII-accelerated atherosclerosis without altering blood pressure and increased vascular expression of Bcl-6, RGS4, and RGS5, which was associated with suppression of inflammatory and atherogenic gene expression in the artery. In vitro studies demonstrated similar changes in AngII-treated macrophages: PPARdelta activation increased both total and free Bcl-6 levels and inhibited AngII activation of MAP kinases, p38, and ERK1/2. These studies uncover crucial proinflammatory mechanisms of AngII and highlight actions of PPARdelta activation to inhibit AngII signaling, which is atheroprotective.

Fig. 1.

PPARδ activation attenuates AngII-accelerated atherosclerosis. Male LDLR^−/− mice were treated with HF/PBS, HF/AngII, or HF/AngII/GW0742 (1 mpk or 10 mpk) for 4 weeks. (A) Aortic PPARδ mRNA expression after 2 and 4 weeks of HF/AngII treatment. Data are mean ± SD (n = 12 per group). *, P < 0.01 vs. HF. (B) Localization of aorta PPARδ protein expression by immunohistochemistry. PPARδ protein expression localized predominantly to macrophage-rich areas (CD68-positive intimal layer) and intimal vascular smooth muscle cells in lesions. (Magnification: ×40.) (C) Representative Sudan IV-stained aortas. (D) Quantification of en face atherosclerotic lesion coverage. Data are mean ± SD (n = 8 per group). *, P < 0.01 vs. HF/AngII by ANOVA.

Fig. 2.

PPARδ activation suppresses AngII-induced vascular inflammation macrophage infiltration. (A) Macrophage abundance (Mac-2 antibody stain) in aortic root lesions. (B) Quantitative analysis of Mac-2-positive area in the aortic root lesions. (C) RT-PCR analysis of aortic expression of two macrophage markers (CD68 and Mac-2). RNA from the whole aortas was analyzed by qRT-PCR and normalized to GAPDH expression. (D) PPARδ agonist inhibits AngII-induced vascular inflammation. RNA levels in the aorta were analyzed by using qRT-PCR and normalized to GAPDH. Data are mean ± SD (n = 12 per group). *, P < 0.01 vs. HF/AngII by ANOVA.

Fig. 3.

AngII suppresses Bcl-6 expression in the aorta and macrophage. RNA isolated from whole aorta (A) and peritoneal macrophages (B) was analyzed by qRT-PCR. Bcl-6 expression was normalized to GAPDH. Data are mean ± SD (n = 6–10 per group). *, P < 0.01 vs. HF/AngII by ANOVA. (C) AngII increases and PPARδ ligand decreases macrophage PPARδ protein expression. (D and E) Bcl-6 protein expression and PPARδ:Bcl-6 interaction in macrophages in response to AngII and/or GW0742. Bcl-6:PPARδ interaction was analyzed by Western blot analyses of total and PPARδ-bound Bcl-6 in macrophage nuclear proteins after pull-down assays. (F and G) MCP-1 and OPN mRNA levels in peritoneal macrophages treated with AngII and/or GW0742. Data are mean ± SD (n = 5 per group). *, P < 0.01 vs. HF/AngII by ANOVA.

Fig. 4.

PPARδ activation inhibits AngII-induced phosphorylation of MAP kinases. Mouse peritoneal macrophages stimulated with AngII in the presence or absence of GW0742 were analyzed by Western blot for MAP kinase activation. Activation of p38 (A) and ERK1/2 (B) was measured by the levels of phosphorylated p38 (pp38) and ERK1/2 (pERK1/2) normalized to total p38 and ERK1/2. n = 3 per group. *, P < 0.05 vs. AngII by ANOVA.

Fig. 5.

PPARδ activation inhibits AngII-mediated G protein signaling and suppression of RGS4 and RGS5 in macrophages. AngII infusion inhibits aorta RGS4 (A) and RGS5 (B) expression, and this effect is reversed by GW0742. Aorta RNA was analyzed by qRT-PCR, and RGS4 and RGS5 mRNA levels were normalized to GAPDH. Data are mean ± SD (n = 5 per group). *, P < 0.001 vs. HF/PBS; **, P < 0.05 vs. HF/AngII (by ANOVA).