Enalapril Alone or Co-Administered with Losartan Rescues Cerebrovascular Dysfunction,but not Mnemonic Deficits or Amyloidosis in a Mouse Model of Alzheimer’s Disease

Animals and treatments

Experiments were in compliance with the Canadian Council on Animal Care and were approved by the Animal Ethics Committee of the Montreal Neurological Institute, McGill University. We used approximately equal numbers of male and female wild-type (WT) and littermate heterozygous transgenic APP mice carrying the Swedish (K670N, M671L) and Indiana (V717F) familial AD mutations driven by the platelet-derived growth factor beta (PDGFβ) promoter on a C57BL/6 background (line J20, [33]. Mice were screened for transgene expression by touchdown PCR using tail-extracted DNA [34]. They were housed under a 12 h light-dark cycle, in a room with controlled temperature (23°C) and humidity (50%), with food and tap water ad libitum. Animals received in drinking water the ACEi enalapril maleate salt (3 mg/kg/day, Merck Frosst Canada Ltée, Kirkland, QC, Canada) that does not cross the BBB [35] alone or in combination with the centrally acting ARB losartan (10 mg/kg/day, Merck Frosst Canada Ltée, Kirkland, QC, Canada and Cedarlane, Burlington, ON, Canada) [36] for three months starting at ∼15 months of age (∼18 months at endpoint, aged mice). The doses of losartan (10 mg/kg/day) and enalapril (3 mg/kg/day) were selected based on their equivalent blood pressure-lowering efficacy [37]. Solutions were replaced once or twice per week. Age-matched control WT and APP mice received unmedicated drinking water. Mouse body weights were monitored weekly or monthly (Table 1). Blood pressure was not monitored in these mice based on the reported lack of additive effects in combined ARB and ACEi therapy in lowering blood pressure [31], and concurrent treatment with losartan that showed no hypotensive effects in similarly aged littermate APP mice, as published in an independent study [15].

Morris water maze (MWM)

Following treatment, mice were tested in a modified MWM, as described by deIpolyi and colleagues [38] and validated by us in our previous studies [15, 39]. Briefly, mice were given three days of visible platform training to exclude visual, motor, or motivational deficits. Wall cues were then switched, the platform moved to a different pool sector (the target quadrant) and submerged for five days of hidden-platform training. Daily escape latencies were recorded. To test memory, the platform was removed and mice were allowed a free swim (probe trial), during which percent time spent and distance travelled in the target quadrant were recorded along with swim speed. Probe trials were conducted 2 h after the last hidden-platform session on day 8 (probe 1), and subsequently 48h later (probe 2). Mice were tracked with the 2020 Plus tracking system and data analyzed with the Water 2020 software (Ganz FC62D video camera; HVS Image, Buckingham, UK). Animals were dried under a heating lamp after each trial, and experiments started at the same time every day.

Vascular and brain tissue collection

A subset of mice that underwent cognitive testing in the MWM was euthanized by cervical dislocation, and used for cerebrovascular reactivity studies and western blot analysis. Specifically, the middle cerebral artery (MCA) was removed for reactivity studies (below). Remaining pial vessels (circle of Willis and ramifications) were dissected and frozen on dry ice, along with cortex and hippocampus from one hemisphere and vessels from untreated WT and APP mice. Samples were stored (–80°C) until western blotting. The other hemisphere was immersion-fixed overnight (4°C, 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline, pH = 7.4), cryoprotected, frozen in isopentane, and stored (–80°C) until cutting of 25μm-thick free-floating coronal sections using a freezing microtome. Another subset of animals was intracardially perfused (4% PFA) under deep anesthesia (65 mg/kg sodium pentobarbital, i.p.). Right hemibrains were processed for cutting of 25μm-thick sections as above. Left hemibrains were paraffin-embedded and cut into 5μm-thin microtome sections.

Vascular reactivity

Isolated, pressurized MCA segments were preconstricted with serotonin (2×10^–7 M; Sigma-Aldrich, St-Louis, MO), and tested for dilatations to acetylcholine (ACh; 10^–10–10^–5 M; Sigma-Aldrich) and calcitonin gene-related peptide (CGRP; 10^–10–10^–6 M; American Peptide, Vista, CA). Constriction to endothelin-1 (ET-1; 10^–10–10^–6 M; American Peptide) and passive diameter decrease during nitric oxide synthase (NOS) inhibition with N ^ω -nitro-L-arginine (L-NNA; 10^–5 M; 35min; Sigma-Aldrich) were tested on vessels at basal tone, using on-line videomicroscopy, as detailed before [15, 41]. Percent changes in vessel diameter from basal or pre-constricted tone were plotted as a function of agonist concentration or time course of NOS inhibition. Dose-response curves generated by GraphPad Prism software (version 4, San Diego, CA) provided the best fitted values of maximal response (EAmax) and of the concentration eliciting half of the EAmax (EC₅₀ value or pD₂ = –), which were used to evaluate agonist efficacy and potency, respectively.

Western blot

Tissues were sonicated in Laemmli buffer (62.5mM Tris-HCl, pH = 6.8; 2.35% sodium dodecyl sulphate (SDS); 100 mM DTT; 10% glycerol; 1 mM EDTA and 0.001% bromophenol blue) and protein concentration measured by the method of Lowry. Proteins (∼35 mg from cortex or 6–15μg from vessels) were separated by SDS-gel electrophoresis and transferred to nitrocellulose membranes incubated (1h) in TBST blocking buffer (50 mM Tris-HCl, pH = 7.5; 150 mM NaCl; 0.1% Tween 20) containing 5% skim milk. Tricine or gradient gels were used for separation of Aβ monomers and oligomers according to [42]. Membranes were incubated overnight with either rabbit anti-SOD1, -SOD2 (1:1000; Stressgen, Ann Arbor, MI), -p67^phox (1:200; Abcam, Cambridge, MA), -AT4 (1:500; Chemicon, Temecula, CA), mouse monoclonal anti-AT1 (1:200; [43]), mouse anti-β-actin (1:10000; Sigma-Aldrich), mouse monoclonal anti-β amyloid 1–16 (6E10 antibody that detects Aβ species and full-length APP; 1:1000; Covance, Emeryville, CA). Membranes were further incubated (2 h) with horseradish peroxidase-conjugated secondary antibodies (1:2000; Jackson ImmunoResearch, Westgrove, PA) and proteins visualized with Enhanced ChemiLuminescence (ECL Plus kit; Amersham, Mississauga, ON, Canada) using phosphorImager (Scanner STORM 860; GE Healthcare, Piscataway, NJ). For the Aβ gradient gel, membranes were first incubated with 6E10 antibody and, after protein detection; membranes were scanned and then re-incubated for detection of β-actin and rescanned for quantification. Densitometric quantification was performed with ImageQuant 5.0 (Molecular Dynamics, Sunnyvale, CA) or Scion image software (NIH, Bethesda, MA).

Histochemistry and immunohistochemistry

Fee-floating thick (25μm) sections were stained with 1% thioflavin-S (8 min) to reveal mature, dense core Aβ plaques or were incubated overnight with antibodies against Aβ_1 - 42 (rabbit anti-Aβ_1 - 42 ; BioSource International, Camarillo, CA), followed by species-specific biotinylated IgG, and the avidin-biotin complex. The reaction was visualized with 0.05% 3,3^′-diaminobenzedene-Nickel (Vector Laboratories, Burlingame, CA). Sections were incubated with goat anti-choline acetyltransferase (ChAT; 1:250; Millipore, Temecula, CA) followed by immunodetection as above, or incubated with rabbit anti-glial fibrillary acidic protein (GFAP; 1:1000; DAKO, Mississauga, ON, Canada), followed by donkey anti-rabbit cyanin 2 (Cy2)-conjugated secondary antibody, 1:400; Jackson ImmunoResearch). Sections were observed under light or epifluorescence microscopy (Leitz Aristoplan microscope, Leica, Montréal, QC, Canada) or confocal microscopy (Zeiss LSM 510, Jena, Germany, emission intensity at 543 nm), and pictures acquired with a digital camera (Coolpix 4500; Nikon, Tokyo, Japan). Digital images (two to three sections per mouse, three to five mice per group) taken under the same conditions were analyzed with MetaMorph 6.1r3 software (Universal Imaging, Downingtown, PA).

Statistical analysis

All data are expressed as mean±SEM. Reactivity dose-response curves, as well as MWM latency curves and probe trials were analyzed by repeated-measures one-way or two-way ANOVA followed by Newman-Keuls post-hoc tests. For immunohistochemical stainings, the areas of interest (somatosensory/cingulate cortex, hippocampus) containing thioflavin-S-, Aβ- or GFAP-positive elements were manually outlined in low-power images, while high-power microscope images of layers II to IV of the somatosensory cortex were used for quantification of ChAT-immunoreactive fibers. Number and/or area occupied by thioflavin-S- or Aβ-positive plaques, GFAP-positive astrocytes and ChAT-positive cholinergic fires was quantified and expressed as number or surface area occupied in the delineated areas of interest. Student t-test was used for two-group comparisons. Statistical analyses were performed with Statistica 10 (Statsoft, Tulsa, OK). P <  0.05 was considered significant.

Enalapril either alone or co-administered with losartan normalized cerebrovascular reactivity by inhibiting oxidative stress

The significantly decreased ability of cerebral arteries to dilate in response to ACh and CGRP in aged APP mice (∼50%) [44, 45] (Fig. 1, Table 2) was completely restored by treatments with enalapril alone and enalapril combined with losartan (Fig. 1, Table 2), confirming the involvement of RAS or of other AT1R-mediated mechanisms in the cerebrovascular dysfunction of APP mice. Deficits were not due to desensitization of cerebrovascular receptors since pD₂ values were comparable between WT and APP arteries in most cases (Table 2). However, in APP mice enalapril slightly increased the ACh pD₂ values, raising the possibility that part of the recovery occurred through sensitization of vasodilatory muscarinic ACh receptors (Table 2). As expected, we found no contractile deficits to ET-1-mediated contractions in aged APP mice (Table 2). Yet, enalapril significantly decreased this response in both aged WT and APP mice without altering ET-1 affinity at contractile receptors (Table 2). The constitutive synthesis and release, or bioavailability, of endothelial nitric oxide (NO), which is essential to maintain basal vessel tone, was unaltered in APP arteries in response to L-NNA, and the treatments had no effect (Table 2).

Both enalapril alone and in combination with losartan significantly reversed the p67^phox upregulated protein levels in pial vessels (Fig. 2). This implicated the p67^phox-containing NADPH oxidase in the dilatory impairments of APP mice, along with its product superoxide known to upregulate SOD2 [46, 47]. The combined treatment, but not enalapril alone, also normalized SOD2 protein levels (Fig. 2), confirming the potent antioxidant effects of losartan [15]. Both treatments induced trends towards a decrease in cerebrovascular SOD1 protein levels (Fig. 2). Interestingly, despite restored cerebrovascular dilatations in aged APP mice, the treatments did not reduce the increased levels of cerebrovascular AT1R in APP mice, although there was a slight reduction in vessels from mice that received the combined treatment (p = 0.053, Fig. 2).

Enalapril either alone or co-administered with losartan failed to rescue memory, cortical AT4R levels, and cholinergic deficits.

As expected from previous studies [15, 45], aged APP mice exhibited severe and highly significant learning and memory deficits, including long-term memory measured 2 days after completing the spatial learning training session in the MWM (day 10, Fig. 3). These behavioral alterations were not reversed by enalapril alone or when co-administered with losartan (Fig. 3) despite a slight, not significant, improvement in the memory performance on the first probe trial on day 8. In addition, none of the treatments restored the significantly reduced neocortical levels of AT4R (Fig. 2) or density of ChAT-immunopositive fibers (–49.1%, p <  0.01) that was slightly, albeit not significantly, increased by both treatments (Fig. 4), this reduced density reflecting the cholinergic deficit reported in APP mice [44, 45] and AD patients [48].

Combined therapy with enalapril and losartan, but not enalapril alone, significantly attenuated astrogliosis but did not reduce the Aβ burden

APP mice exhibited increased reactive astrocytes exemplified by the enhanced GFAP immunostaining in both cortical and hippocampal areas relative to WT littermates (Fig. 5), characteristic of AD brains and APP mice [49]. This activation was significantly reduced by the co-administration of enalapril and losartan, but not by enalapril alone as quantified in the cortex (Fig. 5), suggesting beneficial effects attributed to losartan. Finally, whether alone or co-administered with losartan, enalapril did not reduce the high levels of brain Aβ_1 - 42 monomer (4-kDa band) and oligomers (9- and 56-kDa bands) detected by western blot analysis (Fig. 6). However, there was a significant increase in the cortical and hippocampal surface area occupied by dense core thioflavin-S-positive Aβ plaques in animals under combined therapy (Fig. 6), a tendency that did not reach significance when total Aβ_1 - 42 plaque burden was analyzed (Fig. 6).

Cerebrovascular reactivity and the NADPH oxidase subunit p67^phox

Previous immunohistochemical analyses of the brains of AD patients or AD animal models have shown increased levels of AngII, ACE, or AT1R compared with control subjects [4, 50]. Such increases suggest a role of RAS components in the AD pathology. A possible mechanism is through the interaction of AngII with AT1R that triggers an oxidative stress cascade involving the production of superoxide through NADPH oxidase [51]. Several reports collectively support such a mechanism. The ACEi enalapril blocked the production of AngII in microvessels [52], and the AT1R antagonist losartan reduced NADPH oxidase p67^phox subunit levels in pial vessels [15], as it did for the p47^phox NADPH oxidase subunit in major arteries of spontaneously hypertensive rats [53]. Furthermore, the loss of cerebrovascular dilatory capacity associated with aging was rescued in AT1R-deficient mice [54]. Therefore, our findings indicate that enalapril therapy alone or combined with losartan countered the AngII/AT1R/NADPH oxidase/superoxide deleterious cascade in brain vessels, providing a molecular substrate for the improvement of cerebrovascular function through inhibition of oxidative stress, as documented in previous studies [15, 55].

Treatment benefits on cerebrovascular AT1R and SOD2 levels, and on those of brain GFAP

The upregulation of SOD2 in APP mice compared with WT likely signaled an increase in vascular superoxide levels. Indeed, superoxide and various cytokines are potent inducers of SOD2 [46]. The increased SOD1 and SOD2 levels in APP mouse brain vessels may have been an attempt to safeguard basal NO levels from superoxide, and this might explain the preserved L-NNA responses (Table 2). Free radicals like superoxide sequester vasodilatory NO and disrupt vascular channels and receptors involved in dilatory function [44, 57].

The fact that the combined treatment not only decreased p67^phox levels, but also counteracted cerebrovascular SOD2 upregulation and, slightly albeit not significantly AT1R, and brain astrogliosis in APP mice suggests strongly that these additional effects were attributed to losartan given that enalapril cannot cross the BBB [35], restricting its effects on the luminal side of the vessels. Indeed, we have previously shown that losartan alone normalized the increased protein levels of cerebrovascular SOD2, AT1R and brain GFAP in APP mice [15]. Others have shown normalization of blood levels of IL-1β in APP/PS1 mice [58] treated with losartan. Hence, the attenuation of astrogliosis by losartan may be indicative of improved astrocytic function, and subdued astroglial release of inflammatory mediators, as reported elsewhere [59] and, consequently, an attenuation of oxidative stress [60].

Treatment effects on AT4Rs, cortical Aβ burden, cholinergic and cognitive deficits

Previous data have claimed ACE benefits in the degradation of Aβ peptide in AD [61, 62] and argued that the inhibition of this enzyme may eventually prove counterproductive when administered over prolonged periods [14, 63]. In addition, a prospective cohort analysis has reported lower risk of incident dementia and AD and lower risk of admission to a nursing home and death for AD patients [64] with ARB compared to ACEi, suggesting the superiority of ARB over ACEi and other antihypertensive drugs in reducing the incidence of AD [64]. The lack of cognitive recovery and inability to rescue the cortical cholinergic deficit or AT4R levels observed with enalapril treatment is most likely due by its inability to cross the BBB [35] since brain penetrant ACEi have restored cognition in mice injected with Aβ_25 - 35 [18] or Aβ_1 - 40 [19]. In agreement with our previous study, losartan alone was likewise unable to restore the density of cortical ChAT-positive fibers, and we cannot eliminate the possibility of inefficacy due to the transgenic animal model used [15].

Whereas the failure of the combined enalapril and losartan treatment to rescue cognitive deficits was unexpected, Sink and colleagues [23] found in old hypertensive patients that non-centrally active ACEi were associated with a greater risk for incident dementia. It is thus possible that enalapril, being a non brain-penetrant ACEi, counteracted the beneficial effects of losartan; suggesting that detrimental mnemonic capacities observed in hypertensive patients with non-penetrant ACEi [23] can be recapitulated in animal models of AD. Indeed, we recently found memory improvement along with the normalization of brain AT4R in similarly aged APP mice undergoing a similar curative treatment regimen with losartan [15]. Whether the different findings can be ascribed to differences in the severity of the impairment or the pathology between the cohorts of APP mice, however, cannot be discounted. Nevertheless, the data do not rule out a possible beneficial role for chronic AT1R blockade, which favors the rapid conversion of AngII to its active metabolite AngIV that binds with high affinity to AT4R heavily distributed in neurons of cognitive-processing areas, and previously shown to facilitate learning and memory [20, 66], and to enhance LTP in hippocampal CA1 neurons [67]. A possible reason that the combined therapy was inefficient is the potential inhibition of AngII synthesis by enalapril, which may sufficiently impair the conversion of AngII to the memory-permissive AngIV peptide. Measurements of AngIV levels with enalapril treatment could clarify this hypothesis. Another eventuality to investigate is the potential regulatory relationship that could exist between peripheral and central AngII in AD brain, as recent findings highlight the capacity of peripheral AngII to pass the BBB [68, 69]. Together these finding may support the preferred use of BBB penetrant ACEi when combined with ARB medication for the treatment of AD with ARB medication [13].

Another intriguing finding was the worsening of the dense core Aβ pathology in combination-treated APP mice. Indeed, although previous studies reported that losartan does not affect Aβ burden [16], we found a tendency, though not statistically significant, for losartan to promote the Aβ plaque load in both adult and aged APP mice when administered alone [15]. Other studies with losartan or other ARB have yielded conflicting data on effects of treatment on amyloidosis, varying from no effect in young AD mice treated for one or two months [70, 71] to significant reductions in soluble or deposited Aβ in adult AD mice after several months of treatment (two to five months) [58]. Given these discrepancies, and evidence that the co-administration of enalapril and losartan may affect the endothelial transporters that regulate Aβ influx and efflux to the brain [72], it appears important to further evaluate treatment effects on the Aβ pathology when considering combined ACEi and ARB therapy in patients with AD.

Limitations of the study

Our findings of cognitive benefits with a comparable regimen of losartan in similarly aged APP mice [15], prompted us to test whether its benefits would be enhanced or dampened when combined with an ACEi like enalapril. One possible limitation of the current study is that we did not perform preventive therapy in younger APP mice that were fully protected from cognitive decline with long-term losartan treatment [15]. However, in patients, long-term treatment co-administration of ARB and non brain-penetrant ACEi was not more effective than ARB monotherapy in reducing blood pressure [31] while increasing the risk for kidney problems [32]. In the present study, we did not measure kidney function and/or morphology. Hence, we have no argument to discard any detrimental contribution on cognitive function that could be related to such complications. As we observed no aggravating effects of the dual therapy over enalapril alone, it would seem that, in line with clinical observations [23], that non brain-penetrant ACEi offer little promise for effective AD therapy.

In conclusion, enalapril and combined enalapril/losartan treatment rescued cerebrovascular dilatory capacity, likely by inhibiting the AngII-induced oxidative stress cascade in the cerebrovasculature. The combined therapy exerted additional effects on cerebrovascular oxidative stress and brain inflammation, normalizing cerebrovascular SOD2 protein levels and reducing astrogliosis. The fact that these improvements occurred at an advanced stage of the pathology could be highly relevant to vascular cognitive impairment and vascular dementia. In the context of AD, however, cerebrovascular benefits must be weighed against possible lack of benefits on the amyloid pathology and, possible counteracting effects of non brain-penetrant ACEi on memory since they have been associated with increasing risk for dementia [23] and, particularly so, when combined with ARB [24]. Thus, additional pharmacological investigations must be undertaken to better understand the influence of the co-administration of RAS blockers for therapeutic strategies against AD [73, 74].