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

Results

Characterization of mice

Body weight was increased in both aged wild-type (WT) and eAPP^−/− mice (P<0.05 vs. respective young mice; Table 1). Blood pressure measurements revealed that systolic blood pressure (SBP), mean blood pressure (MBP), and diastolic blood pressure (DBP) were unchanged in young and aged WT and eAPP^−/− mice (P>0.05; Table 1). Blood glucose was also not different between eAPP^−/− mice and their WT irrespective of age (Table 1). Lipid profile was unchanged in plasma of young WT and eAPP^−/− mice (P>0.05; Table 1). Aging caused a slight but significant increase in circulating levels of cholesterol in WT mice (P<0.05 vs. young WT; Table 1). In contrast, levels of triglycerides were decreased in aged eAPP^−/− mice (P<0.05 vs. young eAPP^−/− mice; Table 1). Plasma levels of norepinephrine were significantly increased in aged WT and eAPP^−/− mice (P<0.05 vs. respective young mice; Table 1). Aging significantly increased plasma levels of sAPPα in both WT and eAPP^−/− mice however, levels of sAPPα remained significantly decreased in eAPP^−/− mice as compared to age-matched WT mice (P<0.05; Table 1). However, plasma levels of amyloid-β 1-40 (Aβ_1-40) were unaltered in young and aged WT and eAPP^−/− mice (P>0.05; Table 1).

Table 1. Characteristics of young and aged eAPP^−/− mice.

Parameters	Young			Aged
	WT littermates	eAPP−/−		WT littermates	eAPP−/−
Body weight (g)	30±1 (10)	29±1 (10)		32±1 (12) *	33±1 (12) *
SBP (mmHg)	121±2 (9)	119±2 (9)		120±2 (9)	121±2 (11)
MBP (mmHg)	93±1 (9)	92±1 (9)		94±2 (9)	95±2 (11)
DBP (mmHg)	79±1 (9)	78±1 (9)		81±2 (9)	82±2 (11)
Glucose (mg/dL)	147±8 (10)	156±11 (10)		133±7 (12)	155±10 (12)
Cholesterol (mg/dL)	63±2 (11)	63±3 (11)		88±14 (11) *	72±3 (11)
HDL (mg/dL)	54± 2 (11)	52± 3 (11)		68±13 (11)	54±4 (11)
Triglycerides (mg/dL)	85± 5 (11)	91± 6 (11)		74±8 (11)	63±5 (11) *
Norepinephrine (pg/mL)	1599±222 (16)	1898±280 (16)		3601±1019 (9) *	3371±399 (7) *
sAPPα (pg/mL)	549±39 (10)	244±27 (10) †		734±72 (12) *	428±55 (12) *†
Aβ1-40 (pg/mL)	90±8 (10)	88±10 (10)		93±11 (8)	92±20 (8)
SBP indicates systolic blood pressure; MBP, mean blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; WT, wild-type. Data are means ± SEM and the numbers of mice are indicated in the parentheses. * P<0.05 vs. young mice of same strain; † P<0.05 vs. age-matched WT littermates (two-way ANOVA followed by Tukey’s HSD test).

APP expression and processing in aorta

Western blot analyses revealed that APP expression was significantly lower (by 27%) in the aorta of young eAPP^−/− mice (P<0.05 vs. young WT; Supplementary Figure 1). Aging caused increase in APP expression in WT mice aortas (P<0.05 vs. young WT; Figure 1A) while APP expression was unchanged in aged eAPP^−/− mice aortas (P>0.05 vs. young eAPP^−/− mice; Figure 1A). Release of sAPPα from the aorta was significantly increased in aged WT mice (P<0.05 vs. young WT mice; Figure 1B). In contrast, sAPPα release was not significantly affected by aging of eAPP^−/− mice (P>0.05 vs. young eAPP^−/− mice; Figure 1B). Release of Aβ_1-40 from the aorta was not altered in young and aged WT and eAPP^−/− mice (P>0.05; Supplementary Figure 2). Moreover, protein expressions of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and beta-site APP cleaving enzyme 1 (BACE1) were unaltered in WT and eAPP^−/− mice irrespective of age (P>0.05; Figures 1C, 1D, respectively).

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, 25^th to 75^th 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).

Vascular contraction responses

Contractions to KCl were significantly increased in aged WT and eAPP^−/− mice aortas as compared to young mice (P<0.05; Table 2) while there were no differences between age-matched WT and eAPP^−/− mice (P>0.05; Table 2).

Table 2. Efficacy and potency of vasoconstrictors in young and aged eAPP^−/− mice aortas.

Parameters	Young			Aged
	WT littermates	eAPP−/−		WT littermates	eAPP−/−
KCl (g):	1.95±0.06 (18)	1.87±0.04 (18)		2.53±0.11 (16) *	2.38±0.08 (16) *
PGF2α:
Emax (%)	139±4 (9)	137±3 (9)		123±3 (8) *	124±4 (8) *
pEC50 (−log mol/L)	5.49±0.03 (9)	5.34±0.04 (9) †		5.69±0.03 (8) *	5.57±0.04 (8) * †
Phenylephrine:
Emax (%)	63±6 (9)	50±5 (9)		41±8 (8) *	33±9 (8)
pEC50 (−log mol/L)	7.03±0.02 (9)	6.98±0.02 (9)		6.74±0.07 (8) *	6.68±0.05 (8) *
Emax indicates maximal efficacy; pEC50, potency; WT, wild-type. Data are means ± SEM and the numbers of mice are indicated in the parentheses. * P<0.05 vs. young mice of same strain; † P<0.05 vs. WT littermates of same age (two-way ANOVA followed by Tukey’s HSD test).

Sensitivity to prostaglandin F_2α (PGF_2α)-induced contractions was slightly lower in aortas of young and aged eAPP^−/− mice (P<0.05 as compared to their respective WT mice; Figures 2A, 2B, respectively; Table 2). In contrast, PGF_2α potency was significantly increased in aged WT and eAPP^−/− mice (P<0.05 vs. respective young mice; Table 2), and efficacy of PGF_2α was significantly decreased in aged WT and eAPP^−/− mice (P<0.05 vs. respective young mice; Table 2).

Figure 2. Concentration-dependent contractions to PGF_2α (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).

While no significant differences between age-matched WT and eAPP^−/− mice were observed, the potency of phenylephrine was reduced in aged WT and eAPP^−/− mice (P<0.05 vs. respective young mice; Table 2; Figure 2C, 2D). Furthermore, the efficacy was significantly reduced in aged WT mice (P<0.05 vs. young WT mice; Table 2) while it tended to be reduced in aged eAPP^−/− mice (P=0.088; Table 2).

Vascular endothelial function

Acetylcholine (ACh)-induced endothelium-dependent relaxations were unaltered in young mice with endothelial-specific APP deletion (P>0.05 vs. young WT littermates; Figure 3A and Table 3). Aging caused a significant impairment to ACh in WT mice (P<0.05 vs. young WT mice; Table 3) while the efficacy was unaltered (Table 3). Furthermore, relaxations to ACh were further decreased in aged eAPP^−/− mice (P<0.05 vs. aged WT mice; Figure 3B and Table 3).

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 PGF_2α. * P>0.05 versus wild-type littermates (ANOVA with Bonferroni's correction).

Table 3. Efficacy and potency of endothelium-dependent relaxations in young and aged eAPP^−/− mice aortas.

Parameters	Young			Aged
	WT littermates	eAPP−/−		WT littermates	eAPP−/−
Emax (%):
ACh	78±2 (9)	76±4 (9)		83±2 (8)	69±5 (8) †
ACh + Indomethacin	81±6 (9)	73±4 (9)		84±2 (7)	70±3 (7) †
pEC50 (−log mol/L):
ACh	7.18±0.04 (9)	7.16±0.05 (9)		6.75±0.01 (8) *	6.61±0.05 (8) * †
ACh + Indomethacin	7.33±0.08 (9)	7.25±0.04 (9)		7.17±0.03 (7) #	7.04±0.06 (7) * #
Emax indicates maximal efficacy; pEC50, potency; WT, wild-type. Data are means ± SEM and the numbers of mice are indicated in the parentheses. * P<0.05 vs. young mice of same strain; † P<0.05 vs. WT littermates of same age (two-way ANOVA followed by Tukey’s HSD test); # P<0.05 vs. without indomethacin treatment (unpaired t-test).

Incubation of aortas with indomethacin did not affect relaxations to ACh in young mice (P>0.05; Figure 3C). In contrast, indomethacin significantly improved endothelium-dependent relaxations to ACh in both aged WT and eAPP^−/− mice (P<0.05 vs. aortas without indomethacin; Table 3). However, the efficacy is still significantly reduced in aged eAPP^−/− mice in the presence of indomethacin (P<0.05 vs. aged WT mice with indomethacin; Figure 3D and Table 3). Blockade of NOS with N^ω-nitro L-arginine methyl ester (L-NAME) in the presence of indomethacin completely blocks relaxations to ACh in both young and aged WT and eAPP^−/− aortas (Supplementary Figure 3).

Ex-vivo production of prostaglandins in aorta

We next determined whether age-related changes in vascular prostaglandins contribute to the observed decline in endothelial function of aortas, we measured release of prostaglandins in conditioned medium. Thromboxane B₂ (TXB₂) and prostaglandin E₂ (PGE₂) productions were significantly increased in both aged WT and eAPP^−/− mice (P<0.05 vs. young mice; Figure 4A, 4B) while production of PGF_2α was significantly increased only in aged WT mice (P<0.05 vs. young WT; Figure 4C). In contrast, production of 6-keto PGF_1α (a stable metabolite of prostaglandin I₂) was not different among young and aged WT and eAPP^−/− mice (P>0.05; Figure 4D).

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); PGF_2α (C; n=8 per group for young mice and n=8 per group for aged mice); and 6-keto PGF_1α (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, 25^th to 75^th percentiles, and min-max range. * P<0.05 versus young mice of same strain (two-way ANOVA followed by Tukey’s HSD test).

We also performed western blot analyses for protein expression of COX isoforms in aortas. COX1 expression was not different between WT and eAPP^−/− mice irrespective of age (P>0.05; Figure 5A). In contrast, protein expression of COX2 was significantly increased in both aged WT and eAPP^−/− mice (P<0.05 vs. young mice; Figure 5B). Moreover, the increase in COX2 was significantly reduced in aged eAPP^−/− mice (P<0.05 vs. aged WT mice; Figure 5B).

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, 25^th to 75^th 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).

Protein expression of eNOS

Expression of eNOS was significantly decreased in the aorta of aged eAPP^−/− mice (P<0.05 vs. young eAPP^−/− mice; Figure 6). Furthermore, eNOS protein tended to be reduced in aged WT mice but it did not reach statistical significance (Figure 6).

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, 25^th to 75^th percentiles, and min-max range. * P<0.05 versus young mice of same strain (two-way ANOVA followed by Tukey’s HSD test).

Superoxide anion

To investigate whether endothelial dysfunction in aging was caused by increased superoxide anion production in aorta, we performed HPLC-based assay of 2-hydroxyethidium. Levels of superoxide anion were significantly enhanced in aged eAPP^−/− mice (P<0.05 vs. young eAPP^−/− mice; Figure 7) while superoxide anion levels were unaltered in aged WT mice (P>0.05 vs. young WT mice; Figure 7).

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, 25^th to 75^th 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).

Intracellular cGMP levels

Under basal conditions, cyclic guanosine 3',5'-monophosphate (cGMP) were unchanged in aortas of WT and eAPP^−/− mice irrespective of age (P>0.05; Figure 8). ACh stimulated cGMP levels were significantly increased in young and aged WT and eAPP^−/− mice (P<0.05 vs. basal levels; Figure 8). However, ACh stimulated cGMP levels were significantly decreased in aged WT and eAPP^−/− mice (P<0.05 young mice in the presence of ACh; Figure 8). In contrast, cGMP levels did not differ between WT and eAPP^−/− mice groups under basal and stimulated conditions (P>0.05; Figure 8).

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, 25^th to 75^th 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).

Ex-vivo studies with cytokines

We next attempted in different experimental conditions to provide insight into the effects of APP deletion on eNOS expression in vascular endothelium. Young WT and eAPP^−/− mice aortas were used to examine the effects of pro-inflammatory cytokines cocktail on expression of APP and eNOS. Western blot analysis revealed that APP protein was significantly increased in young WT mice treated with cytokines (P<0.05 vs. PBS treatment; Figure 9A) while APP expression was not significantly affected in young eAPP^−/− mice (P>0.05; Figure 9A). In contrast, eNOS expression was significantly reduced in young eAPP^−/− but not in young WT aortas after cytokines treatment (P<0.05 vs. PBS treatment; Figure 9B). Expressions of iNOS and COX2 were equally increased in both groups of mice (P<0.05 vs. PBS treatment; Supplementary Figure 4).

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, 25^th to 75^th percentiles, and min-max range. * P<0.05 versus control PBS treatment (two-way ANOVA followed by Tukey’s HSD test).

Discussion

There are several major findings in this study. First, protein expression of endothelial APP and production of sAPPα in endothelium of aged WT mice are increased as compared to aged eAPP^−/− mice. Second, impairment of endothelium-dependent relaxations to acetylcholine induced by aging is exacerbated in eAPP^−/− mice aortas. Third, eNOS expression was decreased whereas levels of superoxide anion were enhanced in aortas of aged eAPP^−/− mice. Fourth, pro-inflammatory cytokines increased APP protein expression only in isolated aortas of young WT mice. In contrast, pro-inflammatory cytokines decreased expression of eNOS exclusively in young eAPP^−/− mice. Taken together, the results suggest that during aging endothelial APP exerts vascular protective effects. These effects appear to be mediated by preservation of eNOS and endothelium-dependent vasodilator function.

In the present study we demonstrated for the first time that APP expression and sAPPα release were increased in aged wild-type mice aortas. Importantly, these age-induced changes in expression and processing of APP were not observed in the aorta of eAPP^−/− mice thus demonstrating that endothelial cells are a major site of increased APP expression and production of sAPPα. Of note, Austin and colleagues reported that APP expression is increased mainly in intimal layer of both human and apolipoprotein E-deficient mice atherosclerotic aortas [21]. Importantly, protein expression of mature α-secretase ADAM10 was unchanged in aged aortas. Whether enhanced α-processing of APP could be explained by the increased enzymatic activity of ADAM10, or some other α-secretases, remains to be determined [27].

Prior study reported that hypertension causes shift towards amyloidogenic APP β-processing and development of cerebral amyloid angiopathy [31]. However, we detect neither blood pressure increases nor changes of aortic protein expression of β-secretase, BACE1, and production of Aβ_1-40 in aged wild-type and eAPP^−/− mice indicating that amyloidogenic processing of APP in aorta is not affected by aging. Thus, our study demonstrates that upregulation of APP expression in aged wild-type mice is not induced by alterations of arterial blood pressure.

It is not clear what are the exact mechanisms responsible for increased expression and α-processing of APP in large conduit arteries of aged WT mice. Relevant to our observations, it is well known that aging is characterized by a state of chronic, low-grade inflammation [32, 33]. Moreover, expression of inflammatory cytokines such as TNFα, INFγ, and/or IL-1ß have been reported to be elevated in arteries of aged mice [34–37]. We speculate that the presence of proinflammatory stimuli is the most likely explanation for increased expression and non-amyloidogenic processing of APP. Consistent with this hypothesis, TNFα and IL-1ß increase APP expression and secretion of sAPPα from endothelial cells [19–21, 38]. In the present study, we also demonstrated that ex-vivo treatment of isolated aortas with cocktail of inflammatory cytokines led to increased APP expression in young WT mice but not in eAPP^−/− mice again indicating that endothelium is a major source of APP. The concentrations of cytokines used for ex-vivo studies were higher than published serum cytokines levels detected in-vivo in young control mice [39]. Whether aging significantly increases circulating levels of cytokines tested in our study remains to be determined. Nevertheless, it is conceivable that the observed upregulation of APP represents an adaptative/protective response of endothelial cells to aging-induced inflammation [40, 41].

Although plasma levels of sAPPα were lower in eAPP^−/− mice as compared to WT mice, aging increased sAPPα levels not only in WT mice but also in eAPP^−/− mice. This indicates that other cell types besides endothelial cells must contribute to elevation of circulating sAPPα. It is well known that APP is also expressed in platelets and cleaved by non-amyloidogenic APP processing thereby releasing sAPPα upon activation [38, 42, 43]. Notably, platelets count is significantly increased in aged wild-type C57BL/6J mice [44]. Increased release of sAPPα from platelets may help explain elevated circulating levels of sAPPα in aged eAPP^−/− mice.

Circulating levels norepinephrine were increased in both aged WT and eAPP^−/− mice despite normal blood pressure. This may explain the observed reduced responsiveness to phenylephrine in aging. Chronic exposure of the peripheral vasculature to high circulating levels of norepinephrine causes compensatory downregulation of reactivity to phenylephrine. Indeed, similar observations were made in healthy human subjects showing that α-adrenergic vasocontraction responsiveness to increased circulating norepinephrine levels is reduced with age in healthy men [45].

Aging also alters arachidonic acid metabolism in endothelial cells and these alterations can profoundly affect vasomotor function [46, 47]. Human and animal studies have established that enhanced production of vasoconstrictor prostaglandins can impair endothelium-dependent relaxations during aging [8, 10]. In subjects older than 60 years, the treatment with COX-inhibitor, indomethacin, potentiates endothelium-dependent vasodilation to ACh thus demonstrating that vasoconstrictor prostaglandins contribute to impairment of endothelium-dependent relaxations in humans [10]. In line with these studies, we presented evidence that indomethacin significantly improved endothelium-dependent relaxations in response to ACh in both aged WT and eAPP^−/− mice aortas. Consistent with these observations, production of TXA₂ and PGE₂ was significantly increased in both aged WT and eAPP^−/− mice. The reason why the production of PGF_2α was increased only in aged WT littermates is unclear and remains to be determined. However, this could be related to different expression levels of COX2 between aged WT and eAPP^−/− mice (see below). In contrast, the production of 6-keto PGF_1α remained unchanged in aged conduit arteries demonstrating that the metabolism of prostaglandin I₂, which causes direct vasodilation in smooth muscle cells, is unaffected.

Interestingly, our study revealed that protein expression of COX2 was significantly increased in both aged wild-type and eAPP^−/− mice while protein expression of COX1 remained unchanged. Although COX1 primarily contributes to basal vascular production of prostaglandins, COX2 also contributes to the production of prostaglandins [48]. Indeed, it has been reported that COX2 is expressed in endothelial cells and that inhibition of eNOS unmasks the ability of ACh to elicit endothelium-dependent contractions which are sensitive to inhibition of COX2 [49]. Thus, increased production of vasoconstrictor prostaglandins by COX2 appears to be responsible for reduced endothelium-dependent relaxations in aged mice. Notably, this phenomenon was not affected by genetic deletion of APP in endothelium.

To determine the reason why endothelium-dependent relaxations to ACh were still impaired in aged eAPP^−/− mice despite treatment with indomethacin, we studied eNOS protein expression. Interestingly, eNOS protein was decreased in the aorta of aged eAPP^−/− mice but not in young eAPP^−/− mice. In contrast, expression of eNOS is unchanged in aged WT mice aortas and this is in line with the previous reports [50, 51]. Moreover, eNOS expression is not altered in conduit and resistance arteries of different mouse models of cardiovascular disease despite presence of inflammatory cytokines [52–54]. These observations are confirmed and further extended by our ex-vivo studies of isolated aortas treated with cytokines cocktail showing that eNOS expression was downregulated in young eAPP^−/− mice, but not in young WT mice. From these findings we concluded that during aging, the presence of APP in aortic vascular endothelium is essential for normal expression and function of eNOS.

Superoxide anion levels were increased in aged eAPP^-/- aortas but not in aged WT aortas. Increased ROS generation and/or reduced antioxidant capacity could contribute to the increased levels of superoxide anion. However, insufficient NO production in aged eAPP^-/- mice can also lead to enhanced production of superoxide anion thereby further exacerbating impairment of endothelium-dependent relaxations (Figure 10). Indeed, several studies provided evidence that NO inactivated superoxide anion by rapid nonenzymatic reaction with superoxide anion [55–58].

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.

We were unable to observe any changes in superoxide anion production in conduit arteries derived from aged WT mice under basal conditions and this is in line with existing literature [37, 51, 59]. In contrast, several studies reported increased levels of superoxide anion in aged wild-type mice arteries [50, 60, 61]. The reason for this discrepancy is unclear. One possible explanation is that vascular superoxide production is increased in very old (31 months old) wild-type mice [37, 61].

Elevation of aortic cGMP levels induced by ACh are decreased in both aged WT and eAPP^−/− mice as compared to young mice aortas while basal cGMP levels were not different between WT and eAPP^−/− mice. As mentioned earlier, the endothelium is a major source not only of NO but of prostaglandins as well [3]. Relevant to our observations, endogenous prostaglandins have been shown to decrease cGMP levels and to induce contractions in response to ACh in the aorta [62, 63] thus suggesting that exaggerated production of prostaglandins during aging can impair ACh-stimulated production of cGMP. At the present time we do not have an explanation as to why levels of cGMP were not decreased in aged eAPP^−/− mice.

In aggregate, the present study demonstrates that endothelial dysfunction (loss of NO) is more pronounced in aged eAPP^−/− mice than in aged WT littermates. We also provide evidence that the absence of APP in endothelial cells causes downregulation of eNOS expression in eAPP^−/− mice. Our findings support the concept that up-regulation of endothelial APP and production of sAPPα are adaptive mechanisms designed to protect blood vessel wall from detrimental effects of inflammation associated with aging. In this regard, preservation of NO production in endothelium of aged aortas appears to be critically important function of endothelial APP.

Author Contributions

Livius d’Uscio: experimental design, data collection and analysis, and manuscript writing. Zvonimir Katusic: experimental design, data interpretation, and manuscript writing.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Funding

This work was supported by National Institutes of Health R01 grant HL-131515 and by the Mayo Foundation.

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