Activation of PPARδ prevents endothelial dysfunction induced by overexpression of amyloid-β precursor protein

Abstract

Aims: Existing evidence suggests that amyloid-β precursor protein (APP) causes endothelial dysfunction and contributes to pathogenesis of atherosclerosis. In the present study, experiments were designed to: (1) determine the mechanisms underlying endothelial dysfunction and (2) define the effects of peroxisome proliferator-activated receptor delta (PPARδ) ligand on endothelial function in transgenic Tg2576 mice overexpressing mutated human APP.

Methods and results: Confocal microscopy and western blot analyses of wild-type mice aortas provided evidence that APP protein is mainly present in endothelial cells. Overexpression of APP significantly impaired endothelium-dependent relaxations to acetylcholine and phosphorylation of endothelial nitric oxide synthase at Ser(1177) in aortas. HPLC analysis revealed that tetrahydrobiopterin (BH(4)) levels were reduced in Tg2576 mice aortas. This was caused by increased oxidation of BH(4) and reduced expression and activity of GTP-cyclohydrolase I. Furthermore, gp91phox protein expression and superoxide anion production were increased in aortas of Tg2576 mice. This augmented superoxide formation was completely prevented by the NADPH oxidase inhibitor VAS2870. Expression of copper-/zinc-superoxide dismutase (Cu/ZnSOD) and extracellular SOD was downregulated. Treatment with PPARδ ligand GW501516 (2 mg/kg/day) for 14 days significantly increased BH(4) bioavailability and improved endothelium-dependent relaxations in Tg2576 mice aortas. GW501516 also normalized protein expression of gp91(phox) and SODs, thereby reducing production of superoxide anion in the aortas.

Conclusion: Our results suggest that in APP transgenic mice loss of nitric oxide and increased oxidative stress are the major causes of endothelial dysfunction. The vascular protective effects of GW501516 in Tg2576 mice appear to be critically dependent on prevention of superoxide anion production.

Figure 1

(A) Effect of endothelial removal on APP expression in the aortas of wild-type mice. Please note that removal of the endothelium (E−) significantly reduced APP protein expression. eNOS expression is shown to control the experiments. Results are mean ± SEM (n = 4). *P < 0.05 vs. aortas with endothelium (E+). (B) Representative confocal images showing staining of thoracic aorta of wild-type mouse aorta with anti-APP (red) and anti-VE-cadherin (green) demonstrates colocalization of the two proteins mainly in vascular endothelium (yellow). 4′,6′-diamiddino-2-phenylindole dilactate (DAPI) was used to visualize nuclei. Data are representative of at least three independent experiments. L, lumen; A, adventitia. Bar denotes 50 µm.

Figure 2

Protein expression of APP (A), α-secretase ADAM10 (B), β-secretase BACE (C), and γ-secretase nacistrin (D) in aortas of wild-type and Tg2576 mice treated without (−) or with (+) GW501516. The bar graphs indicated the results of the relative densitometry compared with β-actin protein. Plasma levels of Aβ-40 and Aβ-42 are shown in (E) and (F), respectively. Results are mean ± SEM (n = 6). *P < 0.05 vs. wild-type mice (ANOVA with Bonferroni's).

Figure 3

(A) Endothelium-dependent relaxations to acetylcholine and (B) endothelium-independent relaxations to NO-donor DEA-NONOate in aortas of wild-type littermates and Tg2576 mice. All data are shown as mean ± SEM (n = 6–7) and expressed as percent relaxation of the sub-maximal contraction to phenylephrine (1–3 × 10⁻⁷ mol/L). (C) Representative western blot analyses of phosphorylation of eNOS at Ser¹¹⁷⁷ in aortas of wild-type and Tg2576 mice without (−) or with (+) GW501516 treatment. Bar graph indicates the relative densitometry compared with total eNOS proteins. Results are mean ± SEM (n = 5–6). *P < 0.05 vs. wild-type mice; **P < 0.05 vs. transgenic T2576 mice without GW501516 (ANOVA with Bonferroni's).

Figure 4

Tetrahydrobiopterin (BH₄) metabolism and GTP-cyclohydrolase I (GTPCH I) in aortas of wild-type and Tg2576 mice without (−) or with (+) GW501516 treatment. Bar graphs showing levels of BH₄ (A), levels of 7,8-dihydrobiopterin (7,8-BH₂) (B), and BH₄ to 7,8-BH₂ ratio (C). Representative western blot analysis showed GTPCH I protein expression of aortas, and bar graph indicated the results of the relative densitometry when compared with β-actin protein (D). Quantitative analysis of enzymatic activity of GTPCH I (E). Data are means ± SEM (6–10). *P < 0.05 vs. wild-type mice; **P < 0.05 vs. transgenic T2576 mice without GW501516 treatment (ANOVA with Bonferroni's).

Figure 5

Quantitative analysis of intracellular superoxide anion (A) and western blot analysis of gp91^phox protein expression (B) in aortas of wild-type littermates and Tg2576 mice without (−) or with (+) GW501516 treatment. Results are means ± SEM (n = 6–9 for superoxide anion and n = 4–5 for gp91^phox). Effect of NADPH oxidase inhibition with VAS-2870 on superoxide anion levels in wild-type and Tg2576 mice aortas (C). *P < 0.05 vs. wild-type mice; **P < 0.05 vs. transgenic T2576 mice; ***P < 0.05 vs. control aortas (two-way ANOVA).

Figure 6

Representative western blot analysis of EC-SOD (A), CuZnSOD (B), and MnSOD (C) protein expression in aortas of wild-type and Tg2576 mice without (−) or with (+) treatment with GW501516. Bar graphs indicate the results of the relative densitometry when compared with β-actin protein. Results are mean ± SEM (n = 6–8). *P < 0.05 vs. wild-type mice; **P < 0.05 vs. transgenic T2576 mice without GW501516 (ANOVA with Bonferroni's).