Mechanisms of vascular dysfunction in mice with endothelium-specific deletion of the PPAR-δ gene

Abstract

Peroxisome proliferator-activated receptor (PPAR)-δ is a nuclear hormone receptor that is mainly involved in lipid metabolism. Recent studies have suggested that PPAR-δ agonists exert vascular protective effects. The present study was designed to characterize vascular function in mice with genetic inactivation of PPAR-δ in the endothelium. Mice with vascular endothelial cell-specific deletion of the PPAR-δ gene (ePPARδ(-/-) mice) were generated using loxP/Cre technology. ePPARδ(-/-) mice were normotensive and did not display any sign of metabolic syndrome. Endothelium-dependent relaxations to ACh and endothelium-independent relaxations to the nitric oxide (NO) donor diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate were both significantly impaired in the aorta and carotid arteries of ePPARδ(-/-) mice (P < 0.05). In ePPARδ(-/-) mouse aortas, phosphorylation of endothelial NO synthase at Ser(1177) was significantly decreased (P < 0.05). However, basal levels of cGMP were unexpectedly increased (P < 0.05). Enzymatic activity of GTP-cyclohydrolase I and tetrahydrobiopterin levels were also enhanced in ePPARδ(-/-) mice (P < 0.05). Most notably, endothelium-specific deletion of the PPAR-δ gene significantly decreased protein expressions of catalase and glutathione peroxidase 1 and resulted in increased levels of H2O2 in the aorta (P < 0.05). In contrast, superoxide anion production was unaltered. Moreover, treatment with catalase prevented the endothelial dysfunction and elevation of cGMP detected in aortas of ePPARδ(-/-) mice. The findings suggest that increased levels of cGMP caused by H2O2 impair vasodilator reactivity to endogenous and exogenous NO. We speculate that chronic elevation of H2O2 predisposes PPAR-δ-deficient arteries to oxidative stress and vascular dysfunction.

Keywords: carotid artery; endothelial dysfunction; endothelial nitric oxide synthase; hydrogen peroxide; nitric oxide; peroxisome proliferator-activated receptor-δ; tetrahydrobiopterin.

Fig. 1.

A: PCR analysis of peroxisome proliferator-activated receptor (PPAR)-δ mRNA in the aorta of wild-type (WT) and endothelium-specific PPAR-δ-deficient mice (ePPARδ^−/− mice). Endothelial cells (ECs) were isolated from mouse aortas and subjected to PCR analysis as described in materials and methods. The remaining aortas without endothelium were also analyzed. The PCR product was 124 bp for the exon 4-deleted PPAR-δ allele. GAPDH was used as a loading control (168 bp). B: endothelial nitric oxide (NO) synthase (eNOS; 62 bp) was used to verify endothelial integrity. Cre⁻, WT littermates; Cre⁺, ePPARδ^−/− mice.

Fig. 2.

Endothelium-dependent relaxations to ACh (A) and endothelium-independent relaxations to the NO donor diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate; B) of right common carotid arteries in WT littermates and ePPARδ^−/− mice. Please note that relaxations to ACh and DEA-NONOate were significantly reduced in ePPARδ^−/− mice compared with WT mice. All data are expressed as percent relaxation of the submaximal contraction to U-46619; n = 6. *P < 0.05 (by ANOVA with Bonferroni's correction).

Fig. 3.

A: endothelium-dependent relaxations to ACh in isolated aortic rings of WT littermates and ePPARδ^−/− mice. B: effect of catalase (1,400 U/ml) on relaxations to ACh in WT mouse aortas. C: effect of catalase (1,400 U/ml) incubation on relaxations to ACh in aortas of ePPARδ^−/− mice. All data are expressed as percent relaxation of the submaximal contraction to phenylephrine; n = 6. *P < 0.05 WT mice; †P < 0.05 vs. ePPARδ^−/− mice (by ANOVA with Bonferroni's correction).

Fig. 4.

Relaxations to the NO donor DEA-NONOate in isolated aortic rings of WT littermates and ePPARδ^−/− mice. A: relaxations to DEA-NONOate were significantly reduced in ePPARδ^−/− mice compared with WT mice (n = 6). *P < 0.05 (by ANOVA with Bonferroni's correction). B: removal of ECs (E−) abolished the difference (n = 6). All data are expressed as percent relaxation of the submaximal contraction to phenylephrine.

Fig. 5.

Top: representative Western blot analysis of protein expressions of phosphorylated (p-)eNOS at Ser¹¹⁷⁷ in aortas of WT and ePPARδ^−/− mice. Bottom: bar graph showing the results of the relative optical densitometry (OD) compared with eNOS protein (n = 8). *P < 0.05 vs. WT mice.

Fig. 6.

Quantitative HPLC analysis of vascular biopterin metabolism in ePPARδ^−/− mice. A–D: bar graphs showing tetrahydrobiopterin levels (A; n = 6), 7,8-diydrobiopterin levels (B; n = 6), the ratio of tetrahydrobiopterin to 7,8-diydrobiopterin (C; n = 6), and the enzymatic activity of GTP-cyclohydrolase I (GTPCH I; D; n = 5). E: GTPCH I protein expression (n = 4) in aortas of WT littermates and ePPARδ^−/− mice. Please note that the bar graph of GTPCH I protein indicates the results of the relative densitometry compared with β-actin protein (F). All results are shown as means ± SE. *P < 0.05 vs. WT mice.

Fig. 7.

Quantitative analysis of superoxide anion (A; n = 5) and H₂O₂ levels (B; n = 8) in aortas of WT littermates and ePPARδ^−/− mice. All results were normalized against tissue protein levels. *P < 0.05 vs. WT mice.

Fig. 8.

A: basal cGMP levels in aortas of WT littermates and ePPARδ^−/− mice (n = 9–11). B: effects of in vitro incubation with catalase (1,400 U/ml) on cGMP levels in ePPARδ^−/− mouse aortas (n = 6). C: effects of H₂O₂ (100 μmol/l) on cGMP levels in WT mouse aortas in the absence or presence of the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 10 μmol/l, n = 5). D: effects of endothelium removal (E−) on cGMP levels in WT mouse aortas in the presence or absence of H₂O₂ (100 μmol/l; n = 7). All results were normalized against tissue protein levels. *P < 0.05 vs. WT mice; † P < 0.05 vs. control ePPARδ^−/− mice; #P < 0.05 vs. H₂O₂.

Fig. 9.

Top: Representative Western blot analysis of CuZnSOD (A), MnSOD (B), and extracellular (ec)SOD (C) protein expression in aortas of WT littermates and ePPARδ^−/− mice. Bottom: bar graphs showing the results of the relative densitometry compared with β-actin protein (n = 8–9).

Fig. 10.

Top: representative Western blot analysis of catalase (A) and glutathione peroxidase 1 (GPx-1; B) protein expression in WT littermates and ePPARδ^−/− mouse aortas in the presence (E+) or absence (E−) of ECs. Please note that removal of the endothelium eliminated the differences between WT and ePPARδ^−/− mice. Successful removal of ECs was confirmed by the absence of eNOS. Bottom: bar graphs showing the results of the relative densitometry compared with β-actin protein. Results are means ± SE; n = 10 for E+ and 4 for E−. *P < 0.05 vs. WT mice; †P < 0.05 vs. E+ (by two-way ANOVA).