Effects of Hypertension and Anti-Hypertensive Treatment on Amyloid-β (Aβ) Plaque Load and Aβ-Synthesizing and Aβ-Degrading Enzymes in Frontal Cortex

Abstract

Epidemiological data associate hypertension with a predisposition to Alzheimer’s disease (AD), and a number of postmortem and in vivo studies also demonstrate that hypertension increases amyloid-β (Aβ) pathology. In contrast, anti-hypertensive medications reportedly improve cognition and decrease the risk of AD, while certain classes of anti-hypertensive drugs are associated with decreased AD-related pathology. We investigated the effects of hypertension and anti-hypertensive treatment on Aβ plaque load in postmortem frontal cortex in AD. Aβ load was significantly increased in hypertensive (n = 20) relative to normotensive cases (n = 62) and was also significantly higher in treated (n = 9) than untreated hypertensives (n = 11). We then looked into mechanisms by which hypertension and treatment might increase Aβ load, focusing on Aβ-synthesizing enzymes, β- and γ-secretase, and Aβ-degrading enzymes, angiotensin-converting enzyme (ACE), insulin-degrading enzyme (IDE) and neprilysin. ACE and IDE protein levels were significantly lower in hypertensive (n = 21) than normotensive cases (n = 64), perhaps translating to decreased Aβ catabolism in hypertensives. ACE level was significantly higher in treated (n = 9) than untreated hypertensives (n = 12), possibly reflecting feedback upregulation of the renin-angiotensin system. Prospective studies in larger cohorts stratified according to anti-hypertensive drug class are needed to confirm these initial findings and to elucidate the interactions between hypertension, anti-hypertensive treatments, and Aβ metabolism.

Keywords

Angiotensin-converting enzyme Alzheimer’s disease amyloid β protein anti-hypertensive β-secretase BACE hypertension insulin-degrading enzyme

INTRODUCTION

Epidemiological studies suggest that midlife hypertension is a risk factor for later development of Alzheimer’s disease (AD), the most common form of dementia in the elderly [1, 2]. Although the mechanistic links between hypertension and AD remain elusive, there is evidence of association between hypertension and the classical neuropathological hallmarks of AD; particularly the accumulation of amyloid-β (Aβ) in plaques. In the Honolulu Heart Program/Honolulu-Asia aging Study cohort, elevated midlife systolic blood pressure (≥160 mm Hg) was associated with a significant increase in the number of neocortical and hippocampal Aβ plaques at postmortem examination, and elevated midlife diastolic blood pressure (≥95 mm Hg) was associated with a higher number of neurofibrillary tangles in the hippocampus [3].Similar associations were reported in other postmortem studies [4, 5]. In living participants with or without dementia, the amount of cerebral Aβ detectable by amyloid positron emission tomography (amyloid-PET) increased with diastolic blood pressure [6] and was significantly greater in cognitively normal hypertensive participants who had at least 1 APOE ɛ4 allele [7], an established risk factor for AD [8, 9]. Plasma Aβ₄₂:Aβ₄₀ ratio (mainly driven by Aβ₄₀ level) correlated with systolic blood pressure [10] and with severity of white matter damage [11], which predicts the incidence [12] and progression of decline in AD [13]. Experimental studies in mice [14 –16] and rats [17] provided further evidence that hypertension promotes Aβ accumulation.

At present the mechanism by which hypertension enhances Aβ accumulation is unknown. Hypertension induced by infusion of angiotensin II (AngII) did not alter the expression of mRNAs encoding multiple proteins involved in Aβ production: The α-secretases Adam9, Adam10, and Adam17, the β-secretases Bace1 and Bace2 and the γ-secretase components Psen1, Psen2, Aph1a, Aph1b, Psenen, and Ncstn [18] in C57/BL6 mice. However, intravenous infusion of AngII enhanced β-secretase cleavage of amyloid-β precursor protein (AβPP) in Tg2576 mice, which overexpress a mutant form of AβPP bearing the Swedish mutation (KM670/671NL) [15]. Intracerebroventricular infusion of AngII in adult wild-type Sprague-Dawley rats increased activity of Aβ synthesizing enzymes and Aβ₄₂ production and also increased tau phosphorylation, and all of which was inhibited by an AngII receptor antagonist, losartan [19, 20]. Similarly AngII exposure increased β-secretase activity in Chinese hamster ovary cells producing Aβ [15].

In recent years, the potential protective effect of anti-hypertensive therapies on cognitive decline and AD has become a topic of increasing interest and a number of reviews have attempted to evaluate the somewhat inconsistent evidence from longitudinal cohort studies and clinical trials [21 –23]. The data are encouraging and discrepancies between the studies mainly concern differential effects of the anti-hypertensive drug classes. Fournier et al. [23] concluded that the angiotensin receptor blockers (ARBs) and dihydropyridine calcium channel blockers (CCBs) have greater protective effects than do angiotensin-converting enzyme inhibitors (ACEIs) and diuretics, and that β-blockers may be neutral or even worsen cognitive decline. The amount of cerebral Aβ detected by amyloid-PET in cognitively normal adults was higher in those with untreated hypertension than in the treated or normotensive participants [7].

There is also evidence from postmortem studies that antihypertensive treatment reduces Aβ plaque pathology [3 , 24]. In one of these studies, Hajjar et al. [24] found less Aβ deposition after treatment with ARBs than other classes of anti-hypertensives or in brain tissue from people with a history of untreated hypertension. The ARBs valsartan [25] and candesartan [26] reduced oligomerization of Aβ in vitro, as did the diuretic furosemide and the CCB nitrendipine [26], while the ARBs valsartan [25] and losartan [27] reduced Aβ plaque formation in transgenic mouse models of Aβ accumulation. The mechanism (s) by which anti-hypertensive drugs might decrease Aβ accumulation are largely unstudied. Paris et al. [28] showed that dihydropyridine CCBs improved Aβ clearance across the blood-brain barrier (BBB) in a mouse model of Aβ accumulation and Wang et al. [25] showed that ARB treatment increased activity of an Aβ-degrading enzyme, insulin-degrading enzyme (IDE).

In this study of postmortem tissue we initially performed a retrospective analysis to investigate whether hypertension and anti-hypertensive treatment was associated with Aβ plaque load in the frontal cortex of a series of AD brains in which the plaque load had been routinely measured. Since there is some evidence for a relationship between APOE genotype and hypertension [29], we included an analysis of APOE ɛ4 allele presence or absence. Our finding in this study of significantly increased Aβ load in the AD cases with a history of hypertension compared to normotensive cases, and in treated than untreated hypertensive cases guided a series of subsequent investigations of possible mechanisms by which hypertension and anti-hypertensive treatment might have increased Aβ production or proteolytic degradation. In particular, we investigated the following with respect to hypertensive status and treatment, in AD and neuropathologically normal controls: Activity of β- and γ-secretases, responsible for sequential cleavage of AβPP to Aβ, and concentration and activity of three Aβ-degrading enzymes, neprilysin (NEP) [30 –32], IDE [33, 34], and angiotensin-converting enzyme (ACE) [35, 36]. Levels of ACE and IDE were decreased in cortex from people with a history of hypertension, suggesting that hypertension may affect Aβ catabolism, and the level of ACE was increased in cases where hypertension was treated, suggesting feedback upregulation of the renin-angiotensin system. Prospective studies in larger cohorts stratified according to anti-hypertensive drug class are needed to confirm these initial findings.

MATERIALS AND METHODS

Brain tissue

Brain tissue was obtained from the South West Dementia Brain Bank (SWDBB), University of Bristol, with research ethics committee approval from NRES Committee South West – Central Bristol. The brains had been divided mid-sagittally at autopsy, the left cerebral hemisphere sliced and frozen at –80°C, and the right hemisphere fixed in 10% buffered formalin for 3 weeks before its embedding for paraffin histology. All cases had been subjected to detailed neuropathological assessment and diagnosis had been made following standard protocols [37, 38]. According to National Institute on Aging-Alzheimer’s Association guidelines, AD neuropathological changes were a sufficient explanation for the dementia in all of the AD cases [37] and the controls had no clinical history of cognitive decline and showed no or minimal AD changes (up to Braak tangle stage II) or other neuropathological abnormalities.

For immunohistochemical analysis we used 7-μm paraffin sections from the right frontal lobe. For biochemical analysis, frozen tissue (200 mg) from left midfrontal cortex (Brodmann area 46) was homogenized in either 0.5% Triton X-100 lysis buffer or 1% sodium dodecyl sulphate (SDS) lysis buffer (see individual assays) in a Precellys 24 homogenizer (Stretton Scientific, Derbyshire, UK) for 2×15 s at 6000× g, centrifuged at 13, 000× g for 15 min at 4°C and stored at –80°C. Total protein was measured using the Total Protein kit (Sigma Aldrich).

Study cohorts

Diagnostic criteria for retrospective diagnosis of hypertension were: Sustained SBP ≥140 mm Hg and/or DBP ≥90 mm Hg recorded on three or more occasions. Cases in which all blood pressure readingsrecorded in the medical notes were within the normal range were classified as normotensive; furthermore these cases had no history of any prescriptions for anti-hypertensive drugs. The hypertensive cases were stratified into those with a history of anti-hypertensive treatment (prescription of one or more anti-hypertensive drugs listed in the medical records) and an untreated group (no history of any anti-hypertensive drug prescriptions). MRC database identifiers, pathological and demographic data and details of measurements available for individual cases are listed in Supplementary Table 1.

Measurement of Aβ plaque load

For initial analysis, we used measurements of frontal Aβ plaque load that had previously been determined in 82 AD brains: 62 cases from normotensive patients and 20 from patients with a history of hypertension. Of the hypertensive cases, only 9 had been treated. Age and postmortem delay did not differ significantly between the groups used for pairwise comparisons. Previous studies by our group found no effect of simulated postmortem delay of up to 72 h on Aβ measurements [39]. Braak tangle stage was not significantly different between groups used for pairwise comparisons; however the normotensive group tended to include more cases with lower Braak tangle pathology (including 3 cases with Braak tangle stage III) (Supplementary Figure 1). Demographic data for this cohort are summarized in Table 1.

The field fraction (percentage area) immunopositive for Aβ (labeled with antibody to Aβ residues 17–24, clone 4G8 from BioLegend, London, UK; 1:16000 overnight incubation after immersion of sections in formic acid, visualized with avidin-biotin horseradish peroxidase complex kit from Vector Laboratories, Burlingame, CA) was measured in 10 areas of cortex covering 4 mm². Histometrix software (Kinetic Imaging, Wirral, UK) driving a Leica DM microscope with a motorized stage was used to make an unbiased selection of the 10 areas, as previously described [40]. Aβ-laden blood vessels were excluded fromanalysis.

Measurement of β-and γ-secretase activity

Retrospective analysis of β-secretase and prospective analysis of γ-secretase activity was performed in 108 brains from AD and neuropathologically normal controls: 77 cases from normotensive patients (50 AD and 27 controls) and 31 from patients with a history of hypertension (18 AD and 13 controls). Frontal Aβ plaque load was available for all AD cases. Of the hypertensive cases, 13 had been treated. Postmortem delay did not differ significantly between groups used for pairwise comparisons but age was significantly higher in the hypertensive cohort. Demographic and neuropathological data for this cohort are summarized in Table 2.

β-secretase activity had been measured previously [31 , 42]. As described, the fluorogenic substrate Mca-SEVNLDAEFRK (Dnp)RR-NH₂ (R&D Systems), containing part of human AβPP sequence modified by the Swedish double mutation, was used according to the manufacturer’s guidelines to measure β-secretase activity in brain homogenates prepared in 0.5% Triton X-100 lysis buffer diluted 1:200 in 0.1 M sodium acetate buffer (pH 4, 37°C). Each homogenate was assayed in duplicate in the absence, and once in the presence, of the β-secretase inhibitor III (Millipore, Durham, UK, 5 mM). Seven two-fold serial dilutions of recombinant human β-secretase (R&D Systems, 20000 –156 ng/ml) were also assayed, in the absence and in the presence of β-secretase inhibitor III, on each plate. After a 3 h incubation at 37°C, fluorescence was measured by excitation at 320 and emission at 405 nm in a FLUOstar Optima plate reader (BMG LABTECH Ltd, Aylesbury, UK). β-secretase-specific activity was determined by subtracting the fluorescent signal after inhibition from that in the paired uninhibited wells. β-secretase activity in relative fluorescence units (rfu) was interpolated from the standard curve and adjusted for total protein content.

γ-secretase activity was measured in the same homogenates. The fluorogenic substrate Nma-GGVVIATVK (Dnp)dRdRdR-NH₂ (Merk Millipore, Darmstadt, Germany), containing the C-terminal AβPP amino acid sequence that is cleaved by γ-secretase, was used according to the manufacturer’s guidelines to measure γ-secretase activity in brain homogenates prepared in 0.5% Triton X-100 lysis buffer. Brain homogenates were diluted 1:50 in 0.1 M sodium acetate buffer (pH 4, 37°C) and seven two-fold serial dilutions of a standard reference brain homogenate were included on each plate. Each sample was assayed in duplicate in the absence, and once in the presence, of two γ-secretase-specific inhibitors, L-685, 458 (Tocris Bioscience, Bristol, UK, 100μM) and DAPT (N-[N- (3, 5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (Enzo Life Sciences, Exeter, UK, 200μM) in black F16 Nunc Maxisorp plates (Fisher Scientific). Following a 2 h incubation at 37°C (with agitation, protected from light), fluorescence was measured by excitation at 360 and emission at 440 nm in a FLUOstar Optima plate reader (BMG LABTECH Ltd). γ-secretase-specific activity was determined by subtracting the fluorescent signal after inhibition from that in the paired uninhibited wells. γ-secretase activity (in rfu) was interpolated from the standard curve and adjusted for total protein content. The mean intra-assay CV was 4.41% (s.d. 3.33) and the inter-assay CV was 7.64% (s.d. 3.66).

Measurement of ACE, IDE, and NEP concentration and activity

Retrospective analysis of ACE, IDE, and NEP concentration and activity was performed in 85 brains from AD and neuropathologically normal controls: 64 cases from normotensive patients (43 AD and 21 controls) and 21 from patients with a history of hypertension (12 AD and 9 controls). Frontal Aβ plaque load was available for all AD cases. Of the hypertensive cases, 9 had been treated. Postmortem delay did not differ significantly between groups. In previous studies by our group ACE, IDE and NEP activities were found to be stable for at least 72 h under conditions of simulated postmortem delay [34, 43]. Age was significantly higher in the hypertensive cohort. Demographic and neuropathological data for this cohort are summarized in Table 3.

ACE, IDE, and NEP protein and activity in these cases had been previously measured and published [30 , 41]. ACE protein had been measured using the ACE Duoset ELISA kit (R&D systems) and ACE activity had been measured using the ACE1-specific fluorogenic substrate Abz-FRK (Dnp)-P (Biomol International, Exeter, UK) in the presence of captopril (1 mM) in brain homogenates prepared in 1% SDS lysis buffer, as detailed in Miners et al. [35, 36]. IDE protein had been measured by sandwich ELISA with rabbit anti-human IDE capture antibody (Abcam, Cambridge, UK) and mouse anti-IDE detection antibody (R&D Systems, Abingdon, UK) in brain homogenates prepared in 1% SDS lysis buffer, and IDE activity had been measured using an immunocapture-based assay with an IDE-specific capture antibody (rabbit polyclonal anti-IDE, Abcam) and the fluorogenic peptide substrate Mca-RPPGFSAFK-OH (R&D Systems) in brain homogenates prepared in 0.5% Triton X-100 lysis buffer, as detailed in Miners et al. [33, 34]. NEPprotein had been measured using the Neprilysin Duoset ELISA kit (R&D systems) in brain homogenates prepared in 1% SDS lysis buffer, and NEP activity had been measured using an immunocapture-based assay with a NEP-specific capture antibody (goat anti-human NEP, R&D Systems) and the fluorogenic peptide substrate Mca-RPPGFSAFK-OH (R&D Systems) in brain homogenates prepared in 0.5% Triton X-100 lysis buffer, as detailed in Miners et al. [30 –32].

Statistical analysis

Pairwise comparisons between groups were made by Mann-Whitney U test. Spearman’s test was used to assess correlations between postmortem delay and all measured proteins (for results see Supplementary Table 2). Post-hoc statistical power calculations were made using G^*Power 3.1.9.2 software [44]. P values <0.05 were considered statistically significant.

RESULTS

Increased frontal Aβ load in AD with history of hypertension

Frontal Aβ load was significantly higher in the hypertensive than normotensive AD cases (p = 0.020; Fig. 1A). Frontal Aβ load tended, although not significantly so, to be higher in treated than untreated hypertensive cases (p = 0.07; Fig. 1B). In this cohort possession of APOE ɛ4 was associated with a non-significantly greater frontal Aβ load. Frontal Aβ load was significantly higher in the ɛ4+ hypertensive than ɛ4+ normotensive cases (p = 0.009) (Supplementary Figure 2).

Aβ-synthesizing enzyme activities are not altered in AD or controls with history of hypertension, or affected by anti-hypertensive treatment

β- and γ-secretase activities in midfrontal cortex were higher in AD than control cases (β-secretase p = 0.031; γ-secretase p = 0.058) (Supplementary Figure 3), therefore analysis of the influence of history of hypertension was performed both with the diagnosis groups combined and also separately in AD and controls. β- and γ-secretase activities were similar in hypertensive and normotensive cases, and in treated and untreated hypertensive cases in all diagnosis groups (Fig. 2).

Hypertension, anti-hypertensive treatment, and Aβ-degrading enzymes

Analysis was performed both with diagnosis groups combined and also separately in AD and controls, as there is evidence that ACE, IDE and NEP are altered in AD [31]. Levels and activities of ACE, IDE and NEP were compared between hypertensive and normotensive groups (Supplementary Table 3). ACE protein was significantly lower in the combined hypertensive group (p = 0.010) and in hypertensive AD cases (p = 0.009) but not hypertensive controls (Fig. 3A–C). Similar but non-significant trends were observed for ACE activity (Fig. 3D–F). IDE protein was significantly lower in the combined hypertensive group (p = 0.036) but not when cases were stratified according to diagnosis. IDE activity was significantly higher in hypertensive controls (p = 0.027) but not in the combined or AD group (Fig. 3G–L). NEP protein and activity did not differ significantly between groups.

Levels and activities of ACE, IDE and NEP were also compared between treated and untreated hypertensive cases (Supplementary Table 4). ACE protein was significantly higher in treated cases with diagnosis groups combined (p = 0.008) and in treated AD cases (p = 0.016), and a similar trend was observed in treated controls (Fig. 4A–C). However, the increases in ACE protein were not reflected in ACE activity (Fig. 4D–F). IDE and NEP protein and activity did not differ significantly between groups. Post-hoc analysis estimating the power of the test to reject a type II error (reject the hypothesis falsely) at the p = 0.05 level for ACE protein between hypertensive and normotensive groups was 70% in the combined cohort and 79% in the AD cohort. For ACE protein between treated and untreated groups the figures were 81% in the combined cohort and 83% in the AD cohort.

DISCUSSION

This retrospective study has shown significantly higher frontal Aβ load in AD cases with a history of hypertension, in keeping with prior evidence from other human postmortem [3, 4] and neuroimaging studies [6, 7], and studies in mice [14 –16] and rats [17]. Although Braak tangle stage did not differ significantly between the hypertensive and normotensive group, the hypertensive group did tend to have slightly more advanced Braak tangle stages, raising the possibility that hypertension may also exacerbate this aspect of AD pathology as well. We have expanded on previous studies by investigating several mechanisms by which hypertension might increase Aβ load, focusing on the synthesis and enzymatic degradation of Aβ.

Our findings indicate that β- and γ-secretase activities are unchanged by hypertensive status, arguing against increased Aβ synthesis as an explanation for increased Aβ load in hypertensives. This is in keeping with a study in mice in which hypertension induced by infusion of AngII had no effect on the expression of AβPP or the secretase enzymes [18], but is in contrast to other AngII infusion studies in mice [15] and rats [20]. Of the three Aβ-degrading enzymes we investigated, the concentration of two—ACE and IDE—was significantly decreased in the frontal cortex in hypertensive cases, particularly those with AD. Although we did not find that the activity of these enzymes was significantly reduced in hypertension, this finding raises the possibility that one contributor to the increased Aβ load in hypertension may be a decrease in Aβ catabolism. Studies that have shown that ACE converts Aβ₄₃ (recently found to be the earliest form of Aβ deposited in a mouse model of AD [45]) to Aβ₄₁ [45], and Aβ₄₂ to the less amyloidogenic form Aβ₄₀ [46, 47].

In this study we examined only a few of the potential contributors to increased accumulation of Aβ in AD. Several other effects of hypertension may also play a role. These could include impaired perivascular drainage as a consequence of collagenous thickening and reduced pulsatility of arteries and arterioles [48 –50]; reduced receptor-mediated transport of Aβ across the endothelium into the bloodstream; degeneration of pericytes [51], leading to reduced clearance of soluble Aβ₄₀ and Aβ₄₂ from the interstitial fluid [52]; altered expression of AβPP [53, 54]; or reduction in the activity of any of several Aβ-degrading enzymes other than those measured in the present study [55 –57].

In previous postmortem [3 , 24] and clinical studies [7], anti-hypertensive treatment was reported to reduce Aβ pathology. Unexpectedly, we found frontal Aβ load to be higher in the treated than untreated hypertensives. One possible explanation is that treatmentwas more likely to be prescribed to people with more marked or more persistent hypertension. However, there were several limitations to the present study that should be considered in interpreting the findings. The cohort size was small, particularly that of the treated hypertensive group, and although post-hoc power analyses indicated reasonable statistical power, this type of power calculation cannot be used to support findings retrospectively. Furthermore, the retrospective nature of the study imposed multiple constraints on the data acquisition and quality assurance: In many cases we lacked information on the duration and effectiveness of anti-hypertensive treatment, the criteria for diagnosis and treatment of hypertension have changed over time, and there was inconsistency in the way in which blood pressure was measured—in some cases automated, in others manual. A final consideration is that the cohort was too small for meaningful stratification of cases according to anti-hypertensive drug class, while polypharmacy (i.e., being on more than one type of blood pressure drug concurrently) is also often common. We noted that 89% of treated cases had received either diuretics or β-blockers, which have little protective effect on cognition and may even exacerbate cognitive decline [23], and 33% were prescribed ACE inhibitors, which were found to have ambiguous effects on cognition [23] and may even increase mortality rate in AD [22]. Only 22% were prescribed a CCB and none were prescribed an ARB, the drug classes shown to have the greatest protective potential against AD [23]. For all of these reasons, and the reported differential effects of various anti-hypertensive drugs on AD, the findings on subgroup analysis in the current study cannot be taken to indicate that Aβ accumulation is increased by treatment of hypertensionper se.

As far as we are aware, this is the first study to investigate the possible influence of anti-hypertensive treatment on the level or activity of Aβ-synthesizing and Aβ-degrading enzymes in human brain tissue. ACE protein level in the frontal cortex was significantly increased in treated hypertensives, particularly in the AD group. While for the reasons discussed above, caution is warranted in interpreting this finding, it raises the possibility that antihypertensive therapy may cause feedback upregulation of ACE production within the brain. Although this is unlikely to be have an impact on the control of blood pressure, it is noteworthy that ACE has a wide range of substrates and probable physiological functions in the CNS [58] that have the potential to be affected if the production of this enzyme is increased.

In conclusion we have shown that hypertension increases plaque-associated Aβ in the frontal cortex. This may be partly attributable to decreased Aβ catabolism but other mechanisms are likely to contribute and warrant further investigation. Prospective studies of larger cohorts are needed to assess the influence of different classes of anti-hypertensive drug on Aβ pathology, and the impact this has on their efficacy in reducing cognitive decline and protecting against AD.

Footnotes

ACKNOWLEDGMENTS

This study was supported by University of Bristol Alumni and Alzheimer’s Research UK. The SWDBB, which provided the tissue and clinical data for this study, is supported by BRACE (Bristol Research into Alzheimer’s and Care of the Elderly), Brains for Dementia Research and the Medical Research Council.

Authors’ disclosures available online ().

Appendix

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