Endothelium-specific deletion of amyloid-β precursor protein exacerbates endothelial dysfunction induced by aging

Figures

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  Figure 1. (A) Effects of aging on protein expression of APP in the aortas of wild-type (WT) littermates and eAPP−/− mice (n=10 per group). (B) Effects of aging on ex-vivo sAPPα secretion from wild-type (WT) littermates and eAPP−/− mice aortas. The supernatants were collected and analyzed for sAPPα levels. Results were normalized against tissue protein levels (n=12 per group for young WT littermates and eAPP−/− mice and n=15 per group for aged WT littermates and eAPP−/− mice). (C) Effects of aging on protein expression of ADAM10 in the aortas of wild-type (WT) littermates and eAPP−/− mice (n=6 per group). (D) Effects of aging on protein expression of BACE1 in the aortas of wild-type (WT) littermates and eAPP−/− mice (n=6 per group). Western blot results are the relative densitometry compared with β-actin protein. All results are representing box plots with whiskers showing the median, 25th to 75th percentiles, and min-max range. * P<0.05 versus young mice of same strain; † P<0.05 versus age-matched WT littermates (two-way ANOVA followed by Tukey’s HSD test).

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  Figure 2. Concentration-dependent contractions to PGF2α (A, B) and phenylephrine (C, D) in isolated aortic rings derived from young and aged eAPP−/− mice and their respective wild-type (WT) littermates. Results are shown as mean ± SEM (n=9 per group for young WT littermates and eAPP−/− mice and n=8 per group for aged WT littermates and eAPP−/− mice) and contractions are expressed as percentage of response to a second KCl (80 mmol/L). No significant differences were detected between WT littermates and eAPP−/− mice at same age (ANOVA with Bonferroni's correction).

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  Figure 3. Endothelium-dependent relaxations to ACh in isolated aorta from young (A; n=9 per group) and aged (B; n=8 per group) wild-type (WT) littermates and eAPP−/− mice aortas. Effects of indomethacin (Indo; 10-5 mol/L) on responses to ACh in young (C; n=9 per group) and aged (D; n=7 per group) WT littermates and eAPP−/− mice aortas. Results are shown as mean ± SEM and expressed as percent relaxation from submaximal contractions induced by PGF2α. * P>0.05 versus wild-type littermates (ANOVA with Bonferroni's correction).

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  Figure 4. Effects of aging on ex-vivo secretion of prostaglandins from wild-type (WT) littermates and eAPP−/− mice aortas. The supernatants were collected and analyzed for TXB2 (A; n=11 per group for young mice and n=9 per group for aged mice), PGE2 (B; n=11 per group for young mice and n=9 per group for aged mice); PGF2α (C; n=8 per group for young mice and n=8 per group for aged mice); and 6-keto PGF1α (D; n=11 per group for young mice and n=7-9 per group for aged mice). All results were normalized against tissue protein levels and are representing box plots with whiskers showing the median, 25th to 75th percentiles, and min-max range. * P<0.05 versus young mice of same strain (two-way ANOVA followed by Tukey’s HSD test).

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  Figure 5. Protein expressions of COX1 (A) and COX2 (B) in the aortas of wild-type (WT) littermates and eAPP−/− mice. Western blot results are the relative densitometry compared with β-actin protein (n=10 per group for COX1 and n=8 per group for COX2). All results are representing box plots with whiskers showing the median, 25th to 75th percentiles, and min-max range. * P<0.05 versus young mice of same strain; † P<0.05 versus age-matched WT littermates (two-way ANOVA followed by Tukey’s HSD test).

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  Figure 6. Effects of aging on protein expression of eNOS in the aortas of wild-type (WT) littermates and eAPP−/− mice. Western blot results are the relative densitometry compared with β-actin protein (n=10 per group). All results are representing box plots with whiskers showing the median, 25th to 75th percentiles, and min-max range. * P<0.05 versus young mice of same strain (two-way ANOVA followed by Tukey’s HSD test).

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  Figure 7. Quantitative HPLC analysis of superoxide anion in aortas from young and aged wild-type (WT) littermates and eAPP−/− mice aortas. All results were normalized against tissue protein levels and are representing box plots with whiskers showing the median, 25th to 75th percentiles, and min-max range (n=8 per group for young WT littermates and eAPP−/− mice and n=10 per group for aged WT littermates and eAPP−/− mice). * P<0.05 versus young mice of same strain; † P<0.05 versus age-matched WT littermates (two-way ANOVA followed by Tukey’s HSD test).

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  Figure 8. Quantitative analysis of cGMP in aortas from young and aged wild-type (WT) littermates aortas (A) and eAPP−/− mice aortas (B) under basal and ACh (10 μM) stimulated conditions. All results were normalized against tissue protein levels and are representing box plots with whiskers showing the median, 25th to 75th percentiles, and min-max range (n=13 per group for young WT littermates and eAPP−/− mice and n=10 per group for aged WT littermates and eAPP−/− mice). * P<0.05 versus basal levels; † P<0.05 versus young WT mice (two-way ANOVA followed by Tukey’s HSD test).

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  Figure 9. Effects of ex-vivo treatment for 24 hours with cytokines cocktail on protein expressions of APP (A) and eNOS (B) in the aortas of young wild-type (WT) littermates and eAPP−/− mice. Western blot results are the relative densitometry compared with β-actin protein (n=9 per group). All results are representing box plots with whiskers showing the median, 25th to 75th percentiles, and min-max range. * P<0.05 versus control PBS treatment (two-way ANOVA followed by Tukey’s HSD test).

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  Figure 10. Schematic summary of the effects of aging on endothelial function in wild-type (WT) mice (A) and endothelium-specific amyloid precursor protein-deficient (eAPP−/−) mice (B). Please note that expression of APP is increased in aged wild-type mice (A) but not in aged eAPP−/− mice (B) aortas. ↑ = increase; ↓ = decrease; ↔ = no change; eNOS = endothelial nitric oxide synthase; ROS = reactive oxygen species; COX2 = cyclooxygenase 2; TXB2 = thromboxane B2; PGE2 = prostaglandin E2; ACh = acetylcholine.