Angiotensin Receptor Blocker Prevented β-Amyloid-Induced Cognitive Impairment Associated With Recovery of Neurovascular Coupling
Recent studies suggest that vascular risk factors play a considerable role in the development of Alzheimer disease. Furthermore, the use of antihypertensive drugs has been suggested to reduce the incidence of dementia, including Alzheimer disease. In this study, we examined the effects of an angiotensin receptor blocker, olmesartan, on β-amyloid-induced cerebrovascular dysfunction and cognitive impairment. Oral administration of a low dose of olmesartan attenuated cerebrovascular dysfunction in young Alzheimer disease-model transgenic mice (APP23 mouse), without a reduction in the brain β-amyloid level. Moreover, treatment of APP23 mice with olmesartan decreased oxidative stress in brain microvessels. Using an acute mouse model induced by ICV administration of β-amyloid 1-40, we assessed the effect of oral administration of olmesartan on spatial learning evaluated with the Morris water maze. Olmesartan significantly improved cognitive function independent of its blood pressure-lowering effect, whereas there was no improvement by other types of antihypertensive drugs (hydralazine and nifedipine). We found that pretreatment with a low dose of olmesartan completely prevented β-amyloid-induced vascular dysregulation and partially attenuated the impairment of hippocampal synaptic plasticity. These findings suggest the possibility that amelioration of cerebrovascular dysfunction with an angiotensin receptor blocker could be a novel therapeutic strategy for the early stage of Alzheimer disease.
Alzheimer disease (AD) is the most common form of dementia. One of the major neuropathological hallmarks of AD is accumulation of β-amyloid peptide (Aβ) in the brain.1 Aβ is a 38- to 43-amino acid peptide produced from amyloid precursor protein (APP) by proteolytic processing. Abnormal accumulation of Aβ-peptide in the brain is associated with a cascade of pathological events, resulting in dementia.1 Recently, alterations in cerebrovascular regulation related to vascular oxidative stress have been implicated in the mechanisms of the early stage of AD.2–4 The strategy for AD treatment is shifting to earlier stage intervention or prevention of this disease. Amelioration of cerebrovascular dysregulation could be a novel therapeutic target.
Importantly, there is a growing body of evidence indicating an association between vascular risk factors and AD.5 Several epidemiological studies have shown that hypertension in midlife is related to the development of AD.5,6 Furthermore, some antihypertensive drugs reduce the incidence of dementia, including AD. For example, in the Systolic Hypertension in Europe Study, active therapy with a calcium channel blocker, nitrendipine, reduced the incidence of AD.7 The Cache County Study of Memory and Aging concluded that the use of antihypertensive medication is associated with a lower incidence of AD.8 On the other hand, previous studies have revealed contributory functions of the renin-angiotensin system (RAS) in the pathogenesis of AD. Savaskan et al9 reported increased immunoreactivity of perivascular angiotensin-converting enzyme (ACE) and angiotensin II surrounding cortical vessels in the AD brain, suggesting RAS activation in the cerebral vessels in AD. Activation of RAS in the AD brain has also been reported by other groups.10,11 These findings suggest that inhibition of brain RAS could be a new therapeutic strategy for AD.12 More recent studies have shown that ACE inhibitors and angiotensin receptor blockers (ARBs) have favorable effects on cognitive function.13–15 Importantly, these studies suggest the possibility that these medications may produce their effects on cognitive function independent of their antihypertensive actions, although the underlying mechanisms are still unknown.
In this study, we investigated the effect of an ARB, olmesartan, on cognitive dysfunction in an AD mouse model, focusing on its effect on cerebrovascular function. Our results suggest that oral administration of olmesartan at a relatively low dosage prevents Aβ-mediated cognitive decline, associated with recovery of neurovascular coupling.
Please see also the online Data Supplement (http://hyper.ahajournals.org).
Animals and Drug Treatment
All of the animal experiments were performed in compliance with the Osaka University School of Medicine Guidelines for the Care and Use of Laboratory Animals. Male ddY mice and transgenic APP23 mice were used for the study. In all of the experiments, mice were analyzed at the age of 12 to 13 weeks. Administration of antihypertensive drugs was started at 8 weeks of age and continued until the end of experiments.
Hippocampus-dependent learning and memory function of the mice were investigated with the Morris water maze task. To assess basal activity of animals, the open-field test was carried out.
Electrophysiological assessment of hippocampal function in Aβ-injected mice was carried out 6 to 7 days after ICV administration. Field excitatory postsynaptic potential was studied in the CA1 region of hippocampal sections.
Measurement of Aβ
Fresh-frozen mouse brain was serially homogenized into detergent-soluble and guanidine HCl-soluble fractions. The amounts of Aβ X-40 and Aβ X-42 in each fraction were determined by BNT-77/BA-27 and BNT-77/BC-05 sandwich ELISAs (Wako Pure Chemical Industries), respectively, according to the manufacturer’s instructions.
Isolation of Brain Microvessels and Immunocytochemical Staining
Mouse brain microvessels were isolated and characterized as detailed in the Online Data Supplement.
Measurement of Reactive Oxygen Species
Reactive oxygen species (ROS) production in brain microvessels was measured by dihydroethidium (DHE) microfluorography (Please see the online Data Supplement for more details).
Cerebral Blood Flow Analysis
Assessment of cerebral blood flow (CBF) and cerebrovascular reactivity was carried out as described previously4 with some modifications. All of the parameters were simultaneously monitored with a computerized data acquisition system (Unique Acquisition software; Unique Medical).
All of the data were expressed as mean±SEM. Comparison of 2 groups was performed by 2-tailed t test for dependent or independent samples, as appropriate. Comparison of means among ≥3 groups was performed by ANOVA or repeated-measure ANOVA followed by the Tukey-Kramer multiple-range test.
Olmesartan Improved Neurovascular Dysfunction and Decreased ROS in Brain Microvessels in Young APP23 Mice
With the view of establishing a new therapeutic strategy to prevent AD, we initially used young APP23 transgenic mice, a well-validated animal model of AD.16 Although APP23 mice at the age of 12 weeks had no detectable amyloid plaque in the brain and did not exhibit apparent impairment of cognitive function, the amount of soluble Aβ was significantly elevated as compared with that in wild-type mice.16 Because neural activity is closely related to CBF, and its regulation during brain activity largely depends on reactivity of cerebral vessels,17 we focused on cerebrovascular regulation. It is reported that Aβ (especially Aβ1-40) disrupts cerebrovascular reactivity, which contributes to cognitive dysfunction.4,18 To evaluate cerebrovascular reactivity, we used an in vivo CBF monitoring system and examined functional hyperemia and CBF autoregulation.
There was no significant difference in body weight and resting CBF after 4 weeks of pretreatment with olmesartan (Figure S1B and S1C, available in the online Data Supplement). Arterial blood gases and pH were in the physiological range and did not differ among the 3 groups (Figure S1A). In this study, to minimize confounding effects of a possible difference in neuronal activation on functional hyperemia, we recorded somatosensory evoked potentials (SEPs) at the site of CBF recording and adjusted the magnitude of whisker stimulation (Figure 1A, left). The stimulation pulse was adjusted to evoke half of the maximum amplitude of SEPs. Functional hyperemia evoked by whisker stimulation was significantly attenuated in young APP23 mice (Figure 1A, right). Olmesartan partially, but significantly, ameliorated Aβ-induced impairment of functional hyperemia (Figure 1A and 1B). Because the neuronal activation evoked by whisker stimulation was comparable among the 3 groups (Figure S1D), altered neuronal activation might not contribute to the difference in functional hyperemia. Moreover, the magnitude of adjusted stimulation on the basis of SEP level and the amplitude of stimulation required for maximum SEP response were not different among the groups (Figure S1E and S1F), which indicates that the neuronal response to whisker stimulation in young APP23 mice was comparable to that in transgene-negative littermates. Moreover, APP23 mice showed profoundly impaired CBF autoregulation compared with wild-type mice, whereas olmesartan significantly improved the impairment in CBF during stepwise hypotension (Figure 1C).
Because Aβ-induced oxidative stress in brain blood vessels is thought to contribute to cerebrovascular dysfunction and cognitive impairment in APP transgenic mice,3,4 we next evaluated the effect of olmesartan on ROS production in brain microvessels of young APP23 mice. Using DHE microfluorography, ROS signals in blood vessels were significantly stronger in young APP23 mice than in wild-type mice (Figure 2A through 2C; P<0.01). Treatment with olmesartan significantly reduced ROS production in brain microvessels of APP23 mice. Especially in capillaries, ROS signals in APP23 mice treated with olmesartan were comparable with those in wild-type mice (Figure 2A and 2C).
To examine whether the decreased ROS production in brain microvessels and improvement of cerebrovascular reactivity were related to a reduction in Aβ load in the brain, we measured brain Aβ in mice with or without olmesartan treatment. Although the levels of Aβ40 and Aβ42 in the APP23 mouse brain were significantly elevated as compared with those in wild-type mice, no significant difference was observed between APP23 mice with or without olmesartan treatment (Figure S2A and S2B). In this early stage of disease, no obvious amyloid plaque was observed in the brains of both groups (Figure S2C). Therefore, the improved cerebrovascular reactivity in APP23 mice treated with olmesartan could not be attributed to reduction in brain Aβ.
Olmesartan Improved Cognitive Dysfunction in Aβ1-40-Injected Mice
Unfortunately, young APP23 mice did not exhibit apparent cognitive dysfunction. Given the favorable effects of olmesartan, we then examined the effects of olmesartan on cognitive dysfunction using an Aβ1-40-injected mouse model, because this model is one of the useful, well-validated animal models of AD.19 The validity of this model as an AD model was confirmed by our recent study.20 Treatment with olmesartan for 4 weeks showed a reduction in systolic blood pressure (Figure S3A and S3B; P<0.01), whereas Aβ injection into the cerebroventricles alone had no effect on blood pressure. In the hidden platform test of the Morris water maze, Aβ1-40 injection alone significantly impaired the performance as compared with injection of control peptide Aβ40-1 (Figure 3A; P<0.05). However, administration of olmesartan at doses of 0.5 and 1.0 mg/kg per day significantly decreased the escape latency as compared with vehicle treatment (Figure 3A). In the visible platform test, there were no significant differences in escape latency among all of the groups, indicating that visual function was not different among them (Figure S3C). In the probe test, Aβ1-40 injection alone resulted in significantly less time in the target quadrant as compared with Aβ40-1 injection (P<0.05), whereas olmesartan significantly attenuated the impairment caused by Aβ1-40 (Figure 3B; P<0.01). These results clearly demonstrated that olmesartan significantly improved Aβ-induced cognitive dysfunction. In contrast, we found that higher doses of olmesartan (3.0 mg and 6.0 mg/kg per day) resulted in no favorable effect on cognition because of excessive hypotension (Figure S4A and S4B).
However, we found that the dose of olmesartan (1.0 mg/kg per day) slightly but significantly reduced blood pressure (Figure S3B), although olmesartan ameliorated cognitive dysfunction in Aβ1-40-injected mice. To exclude the possibility that this favorable effect on cognitive function might depend on its blood pressure-lowering effect, we also examined other antihypertensive drugs with non-RAS mechanisms, such as hydralazine (a nonspecific vasodilator) and nifedipine (a calcium channel blocker). Of importance, hydralazine (30 mg/kg per day) and nifedipine (10 mg/kg per day) did not improve cognitive dysfunction, although these drugs reduced systolic arterial blood pressure to a similar degree compared with olmesartan (1.0 mg/kg per day; Figure S5A). In the hidden platform test (Figure 4A), administration of olmesartan at a dose of 1.0 mg/kg per day significantly ameliorated the impairment of spatial learning caused by Aβ1-40 injection (P<0.01), whereas the escape latency in the hydralazine and nifedipine groups was comparable with that in vehicle-treated mice with Aβ1-40 injection. In the probe test, the olmesartan-treated group showed a significant increase in the number of crossings of the trained platform (Figure S5C) and searched in the target quadrant for a significantly longer time than did the vehicle-treated group (Figures 4B and S5E). In contrast, the performances of hydralazine- and nifedipine-treated mice were comparable with that of vehicle-treated animals (Figures 4B and S5C). There were no significant differences in escape latency in the visible platform test (Figure S5B) and swim speed (Figure S5D) among these groups, indicating that the differences in performance in this test reflected neither an alteration of visual function or swimming ability. Furthermore, we evaluated the basal activity of olmesartan-treated mice in the open-field test and confirmed that they showed exploratory activity comparable with that of vehicle-treated mice (Figure S6A and S6C). In this test, we also observed that olmesartan-treated mice showed more habituation (a decrease in locomotion and rearing score on examination days) than did vehicle-treated mice, suggesting that olmesartan might decrease anxiety or aggressiveness of Aβ1-40-injected mice (Figure S6B and S6D). Taken together, the present data suggest that improvement of Aβ-induced cognitive dysfunction by olmesartan is independent of its blood pressure-lowering effect.
Olmesartan Prevented Impairment of Synaptic Plasticity and Cerebrovascular Dysfunction Induced by Aβ1-40
To elucidate the cellular mechanisms of the improvement in cognitive dysfunction by olmesartan, we further examined hippocampal function as assessed by long-term potentiation (LTP) in Aβ-injected mice with or without olmesartan treatment. LTP recorded at CA1 was significantly suppressed in Aβ1-40-injected mice compared with that in control (Aβ40-1-injected) mice (Figure 5A and 5B). However, pretreatment with olmesartan significantly attenuated the suppression of LTP in Aβ1-40-injected mice (P<0.01).
We then focused on cerebrovascular regulation. Although arterial blood gases and pH were not outside of the physiological range among all of the groups (Table S1), direct topical application of Aβ1-40 onto the mouse somatosensory cortex significantly attenuated the increase in CBF evoked by whisker stimulation (Figure 6A). Pretreatment with olmesartan significantly prevented Aβ-induced attenuation of functional hyperemia (Figure 6A).
We further assessed CBF autoregulation. Under topical application of Aβ1-40, the reduction of CBF in response to stepwise hypotension in the range of 60 to 90 mm Hg was larger than that in control (Aβ40-1+vehicle-treated) mice (Figure 6B), which indicates that cerebrovascular autoregulation was disrupted by Aβ1-40. On the other hand, animals treated with olmesartan showed recovery of impaired autoregulation in the presence of Aβ1-40. It is noteworthy that treatment with olmesartan ameliorated Aβ-induced cerebrovascular dysfunction.
Recent studies have revealed significant contributory functions of RAS in the pathophysiology of AD. Activation of RAS in the AD brain was reported by some groups.9–11 These findings support the attractive hypothesis that inhibition of the brain RAS could be a new therapeutic strategy for AD.12 Indeed, a recent study showed that treatment with brain-penetrating ACE inhibitors slowed the rate of cognitive decline in AD patients in comparison with other antihypertensive medication.21 However, some in vitro studies have suggested that ACE could play an important role in the metabolism of Aβ,22,23 demonstrating that ACE degraded Aβ and ACE inhibition increased Aβ levels. On the other hand, as treatment of Tg2576 mice with an ACE inhibitor promoted Aβ deposition,24 treatment with ACE inhibitors might be a risk factor for AD. This effect of ACE inhibition on Aβ metabolism should be carefully considered, especially in relation to long-term treatment with ACE inhibitors. Additional studies are necessary to determine the effects of ACE inhibitors both on Aβ deposition in the brain and on cognitive function in AD patients.
In contrast, an ARB could be a more appropriate treatment for AD patients with hypertension. Some clinical evidence suggests that ARBs prevent cognitive decline in patients with hypertension, independent of their blood pressure-lowering effect.13,25 Importantly, a recent study demonstrated that an ARB, valsartan, attenuated oligomerization of Aβ into toxic oligomers, and treatment of Tg2576 mice with valsartan reduced amyloid neuropathology.26 Another report also revealed that telmisartan prevented cognitive deficit in an Aβ injection mouse model through angiotensin receptor blockade and activation of peroxisome proliferator-activated receptor-γ.27 However, it is still an enigma regarding whether ARBs improve cognitive dysfunction. The present study demonstrated that oral administration of an ARB, olmesartan, prevented cognitive impairment in a mouse model of AD, associated with amelioration of Aβ-induced cerebrovascular dysfunction. Our findings suggest important novel mechanisms for the improvement of Aβ-mediated cognitive dysfunction by ARBs.
Our present study provides several important observations. First, low doses of olmesartan (≈1.0 mg/kg per day) improved Aβ-induced cognitive dysfunction. However, other antihypertensive drugs (hydralazine and nifedipine) did not, although the Systolic Hypertension in Europe Study showed that a calcium channel blocker significantly reduced the incidence of AD.7 The neuroprotective effect of calcium channel blockers might depend on their blood pressure-lowering effect. The present study strongly suggests that olmesartan ameliorated Aβ-mediated cognitive deficit through mechanisms independent of blood pressure lowering. Second, olmesartan improved neurovascular dysfunction and decreased ROS production in APP23 transgenic mice. Considerable evidence now indicates the importance of cerebrovascular dysfunction in the pathogenesis of AD.17,28,29 Interestingly, neurovascular coupling is reported to be compromised in AD17 by the enhanced generation of superoxide radicals.4 Thus, the beneficial effect of olmesartan on cognitive dysfunction could be attributed to recovery of cerebrovascular function associated with a significant reduction in oxidative stress. Third, olmesartan restored LTP in the hippocampus of Aβ1-40-injected mice and ameliorated the impairment of cerebrovascular regulation induced by Aβ1-40. Brain activity critically depends on a continuous blood supply, and a focal increase in CBF in a functionally activated area (functional hyperemia) supports normal cognitive function.17 It has been reported that synaptic transmission partly depends on the brain microcirculation.30 Therefore, it is possible that improvement of the microcirculation by olmesartan may contribute to the attenuation of Aβ-mediated suppression of LTP. It is also possible that olmesartan may act directly on neurons in the brain and exert a neuroprotective effect. Angiotensin II induces inflammation and ROS production in the brain via the angiotensin II type 1 receptor, which caused neuronal dysfunction. Selective blockade of the angiotensin II type 1 receptor in the brain might abolish these unfavorable effects directly or through the protective effect of angiotensin II type 2 receptor stimulation.
Importantly, improvement in CBF regulation occurred without a reduction of brain Aβ level, implying that olmesartan protects brain vessels from Aβ-mediated oxidative damage without a reduction of the Aβ level. A recent study also showed that a peroxisome proliferator-activated receptor-γ agonist completely restored cerebrovascular function in transgenic APP mice, without a reduction in brain Aβ level.31 Another report demonstrated that aged Nox2-null APP mice show preserved cerebrovascular function and reduced oxidative stress in cerebral vessels and, furthermore, preserved cognitive function without improvement of amyloid neuropathology.3
Overall, the present study demonstrated that low doses of olmesartan (≈1.0 mg/kg per day) significantly ameliorated Aβ-mediated cerebrovascular and cognitive dysfunction. It is noteworthy that the dose for cognitive improvement (0.5 to 1.0 mg/kg per day) in this study was approximately within the equivalent range of the recommended dosage of olmesartan (20 to 40 mg/d) for the treatment of hypertension in humans. This animal study demonstrated the potential therapeutic strategy using ARB, although further studies are necessary to investigate the clinical effects of olmesartan in AD patients.
We thank Matthias Staufenbiel (Novartis Institutes for BioMedical Research, Basel, Switzerland) for providing APP23 mice, Nobuhisa Iwata (RIKEN Brain Science Institute, Saitama, Japan) for helpful discussion.
Sources of Funding
This work was supported in part by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (to R.M.) and Grants-in-Aid from Japan Promotion of Science; the Japanese Ministry of Education, Culture, Sports, Science, and Technology (to R.M. and N.S.); the Japan Science and Technology Agency (to N.S.); the Takeda Science Foundation (to R.M.); Novartis Pharma AG; Chiyoda; and the Kanae Foundation (to N.S.).
- Received June 30, 2009.
- Revision received July 27, 2009.
- Accepted September 10, 2009.
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