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. 2011 Apr 3;21(5):594–605. doi: 10.1111/j.1750-3639.2011.00486.x

Neprilysin Protects against Cerebral Amyloid Angiopathy and Aβ‐Induced Degeneration of Cerebrovascular Smooth Muscle Cells

James Scott Miners 1, Patrick Kehoe 1, Seth Love 1
PMCID: PMC8094282  PMID: 21382117

Abstract

Neprilysin (NEP), which degrades amyloid‐β (Aβ), is expressed by neurons and cerebrovascular smooth muscle cells (CVSMCs). NEP immunolabeling is reduced within cerebral blood vessels of Alzheimer's disease (AD) patients with cerebral amyloid angiopathy (CAA). We have now measured NEP enzyme activity in leptomeningeal and purified cerebral cortical blood vessel preparations from control and AD patients with and without CAA. Measurements were adjusted for smooth muscle actin (SMA) to control for variations in CVSMC content. NEP activity was reduced in CAA, in both controls and AD. In leptomeningeal vessels, NEP activity was related to APOE genotype, being highest in ε2‐positive and lowest in ε4‐positive brains. To assess the role of NEP in protecting CVSMCs from Aβ toxicity, we measured cell death in primary human adult CVSMCs exposed to Aβ1‐40, Aβ1‐42 or Aβ1‐40(Dutch variant). Aβ1‐42 was most cytotoxic to CVSMCs. Aβ1‐42‐mediated cell death was increased following siRNA‐mediated knockdown or thiorphan‐mediated inhibition of NEP activity; conversely Aβ1‐42‐mediated cytotoxicity was reduced by the addition of somatostatin and NEP over‐expression following transfection with NEP cDNA. Our findings suggest that NEP protects CVSMCs from Aβ toxicity and protects cerebral blood vessels from the development and complications of CAA.

Keywords: amyloid β toxicity, CAA, cerebral amyloid angiopathy, neprilysin

INTRODUCTION

The most common form of cerebral amyloid angiopathy (CAA) is characterized by the accumulation of amyloid‐β (Aβ) in the walls of leptomeningeal and cerebral cortical blood vessels 39, 40. In more severe cases, this is associated with the degeneration of cerebrovascular smooth muscle cells (CVSMCs) in the tunica media 26, 56, 61. The prevalence and severity of sporadic CAA increase with age and in Alzheimer's disease (AD) 27, 29, 30. The Aβ that accumulates in CAA is thought to be predominantly, if not exclusively, of neuronal origin 5, 17, 49. The isoform that is most abundant in vessel walls in CAA is Aβ1‐40 (15), but it is unclear whether this is also the isoform of Aβ most toxic to CVSMCs.

Cerebrovascular accumulation of Aβ is likely to result from combinations of increased neuronal production and decreased removal that may differ between individuals. At least some of the removal of Aβ is thought to be mediated by enzymatic degradation. Neprilysin (NEP) is one of the best characterized Aβ‐degrading enzymes. Within the brain, it is expressed both neuronally and in the tunica media of cerebral blood vessels 7, 32.

Several lines of evidence suggest that decreased expression of NEP may contribute to the development of CAA. Immunolabeling of NEP in blood vessels is reduced in CAA 7, 32 in association with APOEε4 (32), a strong genetic risk factor for CAA 8, 16, 38. The loss of NEP in CAA is not simply a consequence of degeneration of CVSMCs; in severe CAA the labeling of NEP is reduced even in those segments of cortical blood vessels that do not show accumulation of Aβ(32). Furthermore, NEP gene silencing in a human amyloid precursor protein (APP) (J9) mouse model resulted in the development of CAA in a gene‐dosage selective manner, suggesting that loss of NEP activity is sufficient to produce CAA (13).

By immunofluorescent labeling of paraffin sections, we previously demonstrated reduced immunolabeling of vascular NEP in CAA‐laden blood vessels, particularly in APOEε4‐positive patients (32). However, it is not currently known whether NEP is enzymatically active within blood vessels and whether the reduction in immunolabeling reflects loss of NEP enzyme activity. To address this, we have now examined the relationship between NEP activity, CAA severity and APOE genotype in two types of vessel‐enriched preparation (leptomeningeal and purified cerebral cortical blood vessels), to minimize potential confounding effects of other potential sources of NEP activity such as neurons, astrocytes and microglia. In addition, we have measured smooth muscle actin (SMA) level, a marker of smooth muscle cells, in the vessel preparations and used this to adjust NEP activity measurements for CVSMC content and preservation. Lastly, to establish whether or not NEP protects CVSMCs against Aβ‐mediated cytotoxicity (and therefore potentially against complications of CAA such as loss of vasomotor function, and cerebral hemorrhage), we have examined Aβ toxicity to CVSMCs in vitro, and assessed the effect of reducing NEP activity by gene knockdown or adding thiorphan, and of increasing NEP activity by adding somatostatin or transfecting the CVSMCs with NEP cDNA.

METHODS

Study cohort

Brain tissue was obtained from the Human Tissue Authority‐licensed South West Dementia Brain Bank, University of Bristol, UK, with Research Ethics Committee approval. All cases had previously undergone detailed neuropathological examination which included immunohistochemistry for Aβ and phospho‐tau. The probability of AD had been assessed according to the criteria of the Consortium to establish a Registry for Alzheimer's disease (CERAD) (14). All AD cases conformed to the National Institute on Aging–Reagan criteria of a high likelihood that the AD pathology accounted for the dementia (20). The controls had no history of cognitive decline, an absence of AD (as defined by CERAD) or other neuropathological abnormalities, and were of Braak tangle stages 0–III (4).

The scoring of the severity of CAA in the AD and control brains was adapted, as previously described 8, 32 from the method of Olichney and colleagues (37): 0 = no CAA, 1 = trace deposition, 2 = circumferential deposition in some vessels, 3 = widespread CAA and 4 = widespread CAA with dyshoric change. For the present study, cases with CAA severity scores of 0–1 were grouped as “absent or mild CAA,” and those with scores of 2–4 as “moderate to severe CAA.” The cohort was subdivided into the following four groups: controls with absent or mild CAA (n = 9, mean age = 84.2 years, SD = 6.82), controls with moderate to severe CAA (n = 6, mean age = 88.0 years, SD = 7.54), AD cases with absent or mild CAA (n = 10, mean age = 79.5 years, SD = 8.11) and AD cases with moderate to severe CAA (n = 9, mean age = 77.6 years, SD = 7.18). APOE genotyping had previously been performed (8) within the cohort according to established methods (60).

Preparation of leptomeningeal homogenates

Leptomeninges (100 mg) were carefully stripped from frozen brain tissue from the frontal (Brodmann area 6) and occipital cortex (Brodmann area 17–18). The meninges were finely chopped and homogenized at 4°C in 250 µL total extraction buffer (TEB) (Millipore, Watford, UK) [containing {4‐[2‐hydroxyethyl]‐1‐piperazineethanesulfonic acid} (HEPES) (pH 7.9), MgCl2, KCl, (Ethylenediaminetetraacetic acid) (EDTA), sucrose, glycerol, sodium deoxycholate, NP‐40, sodium orthovanadate and a proprietary mixture of protease inhibitors]. We used a Precellys 24 automated tissue homogenizer (Stretton Scientific, Derbyshire, UK) with 2.3‐mm metal beads to prepare homogenates, which were spun at 17 000 × g for 15 minutes. The supernatant was aliquoted and frozen at −80°C until used. Total protein concentrations were determined using Total Protein Kit (Sigma Aldrich, Dorset, UK) according to the manufacturer's guidelines.

The remaining pellet was homogenized in 6.25 M guanidine HCl (Sigma Aldrich, Dorset, UK) in 50 mM Tris (pH 8.0), incubated at 26°C for 4 hours and spun at 17 000 × g. The supernatant was stored at −80°C. Measurement of insoluble (guanidine‐extracted) Aβ in leptomeninges was performed using a sandwich Enzyme‐linked immunosorbent assay (ELISA) as previously described 51, 52.

Preparation of cerebral cortical vessel‐enriched homogenates

Tissue homogenates were prepared from frozen tissue samples of frontal and occipital cortex prior to enrichment for cerebral cortical vessels according to the method of Tsuji and co‐workers (48) with slight amendments. Brain tissue (1.5 g) was homogenized in ice‐cold Phosphate buffered saline (PBS) by use of a hand‐held homogenizer (10 up–down strokes), and filtered through a 70‐µm nylon cell strainer (BD Biosciences, Wiltshire, UK) to remove single cells. Examination of a small amount of the retained material (resuspended in PBS and smeared onto glass slides, fixed in alcohol and stained with Harris' hematoxylin and eosin for microscopic examination) revealed good retention of capillaries and arterioles (Supporting Figure S1). The retained material was recovered from the filter, washed twice in ice‐cold PBS and resuspended in 0.25 M sucrose, which was loaded on a discontinuous sucrose gradient (1.5, 1.0 and 0.25 M) and spun at 70 000 × g for 40 minutes at 4°C. The resultant pellet was washed in PBS twice and then homogenized (as for the leptomeninges) in 250 µL TEB, spun at 17 000 × g for 15 minutes at 4°C and the supernatant stored at −80°C until used. Total protein concentrations were determined using Total Protein Kit according to the manufacturer's guidelines.

Measurement of SMA

Western blot analysis of SMA was performed to assess the specificity of the anti‐SMA antibody (Abcam, Cambridge, UK). The blots revealed a single band migrating at the expected molecular weight of 40 kDa, in tissue homogenates of human frontal cortex (Supporting Figure S2). The same antibody was then used in a direct ELISA to measure the SMA level in the vessel‐enriched extracts of cerebral cortex and the leptomeningeal preparations. Costar EIA microplate wells were coated with 10 µL of homogenate for 2 hours, blocked in 1% milk powder for 2 hours and washed five times in PBS/tween 0.05% before being incubated with anti‐SMA (1:500) for 2 hours. After further washes, rabbit‐Horse radish peroxidase (HRP) (Vector Laboratories, Peterborough, UK) diluted 1:100 in PBS/0.01% tween‐20 was added for 20 minutes in the dark. The plates were washed and Tetramethylbenzidine (TMB) substrate added for 3 minutes. In each plate, SMA levels were calibrated against a serial dilution of reference leptomeningeal vessel preparation to adjust for potential plate‐to‐plate variation. Each sample was run in duplicate and the mean determined. The SMA measurements were used to adjust NEP levels and activity according to the formula: NEPadjusted = NEPsample × (SMAmean/SMAsample), where SMAmean was the mean SMA value for all samples.

NEP activity and ELISA measurements

NEP activity was measured by an immunocapture‐based fluorogenic assay specific for NEP (33), as previously used to measure NEP activity in brain tissue homogenates 34, 35. The assay uses a NEP‐specific capture antibody (goat anti‐human NEP, R&D Systems, Oxford, UK) to extract NEP from tissue homogenates prior to incubation with the fluorogenic peptide substrate Mca‐RPPGFSAFK‐OH (R&D Systems). NEP protein level was determined by indirect sandwich ELISA (Neprilysin Duoset ELISA kit, R&D Systems) as previously described (34).

Measurement of insoluble Aβ in leptomeninges

Insoluble Aβ was measured by sandwich ELISA as described previously 51, 52. The measurements were made on the guanidine HCl‐extractable fraction of homogenates of leptomeninges, prepared as above. The ELISA used monoclonal anti‐Aβ (4G8 clone, raised against amino acids 18–22; Millipore, Watford, UK) for the capture step and biotinylated anti‐human Aβ monoclonal antibody (10H3 clone, Thermo Fisher Scientific, Northumberland, UK) for the detection step. The yield from pelleted vessel‐enriched preparations of cerebral cortex after tissue homogenization was insufficient to allow guanidine extraction of Aβ, precluding measurement of insoluble Aβ in these preparations.

Culture of CVSMCs

Human brain cerebrovascular smooth muscle cells (CVSMCs) were obtained from Sciencell, TCS Cellworks, Bucks, UK. These cells were isolated from human brain vascular tissue and had been previously characterized in‐house by immunofluorescent labeling with antibodies to α‐SMA and desmin. They were supplied cryopreserved at secondary culture. CVSMCs were grown in specially formulated vascular smooth muscle cell medium (Sciencell, TCS Cellworks, Bucks, UK) supplemented with 2% foetal bovine serum, 1% proprietary smooth muscle cell growth supplement and 1% penicillin/streptomycin at 37°C in 5% CO2/95% air in poly‐L‐lysine (15 µg/mL in distilled water)‐coated flasks (Sciencell, TCS Cellworks, Bucks, UK). All experiments were carried out using CVSMCs between the 5th–7th passage.

Immunofluorescent labeling of the cultures for SMA (1:100) (Abcam, Cambridge, UK) and NEP (1:20) (Santa Cruz Biotechnology, Tebu Bio, Cambs, UK) with alexa‐fluor 488 (1:500) (Invitrogen, Paisley, UK) showed the expected filamentous pattern of staining of the CVSMCs for SMA and granular immunopositivity for NEP (Supporting Figure S3). Visualization of SMA‐labeled cells at lower magnification (not shown) revealed a homogenous population of SMA‐positive cells.

NEP siRNA knockdown

CVSMCS were grown to 30%–50% confluence. NEP siRNA (10 nM) (Santa Cruz, from Insight Biotechnology, Wembley, UK) diluted in Optimum transfection medium (Invitrogen, Paisley, UK), was conjugated to INTERFERin (Polyplus Transfection, Illkirch, France) according to the manufacturer's guidelines and added to the cells. The culture medium was exchanged for fresh CVSMC complete medium after 4 hours to minimize cellular toxicity. After 4 days the cells were washed in PBS and detached using 3‐mL Accutase (Invitrogen, Paisley, UK). Some cells were pelleted and lysed in 100‐µL nondenaturing CellLyticTM cell lysis buffer according to manufacturer's guidelines (Sigma Aldrich, Dorset, UK) (for NEP ELISA and enzyme activity measurements). Cells from the same experiment, which were not prepared for cell lysates, were seeded overnight in a poly‐L‐lysine coated 96‐well plate for Aβ1‐42 cytotoxicity assays.

NEP transfection of CVSMCs

CVSMCs were grown to 50%–70% confluence. An untagged full‐length cDNA clone of human NEP (MMEL1) cDNA (5 µg) (Origene, Insight Biotechnology, Watford, UK) diluted in 150 mM NaCl was conjugated to jetPEI (Polyplus Transfection, Illkirch, France) according to the manufacturer's guidelines and added to the cells. The culture medium was exchanged for fresh CVSMC complete medium after 4 hours to minimize cellular toxicity. After 24 hours, the cells were washed in PBS; 3 mL Accutase (Invitrogen, Paisley, UK) was added to detach the cells, which were then pelleted and lysed in 100‐µL nondenaturing CellLyticTM cell lysis buffer according to the manufacturer's guidelines (Sigma Aldrich, Dorset, UK) (for NEP ELISA and enzyme activity measurements). Cells from the same experiment, which were not prepared for cell lysates, were seeded overnight in a poly‐L‐lysine coated 96‐well plate and underwent Aβ1‐42 cytotoxicity assays.

Aβ cytotoxicity assay

CVSMCs (7500/well) were added to poly‐L‐lysine coated 96‐well plates and left overnight to achieve 70%–90% confluence. The culture medium was replaced by a serum‐free medium (complete CVSMC medium in the absence of fetal bovine serum), and 4 hours later we added 2.5, 5 or 10 µM Aβ1‐40, Aβ1‐42, Aβ1‐40(Dutch variant) or peptide with a scrambled sequence of Aβ1‐42 amino acids (Aβ1‐42scrambled) (Anaspec, Tebu Bio, Cambs, UK) that had been diluted in the serum‐free medium and allowed to oligomerize overnight at room temperature before use (50). Control CVSMCs were left in serum‐free medium without the addition of Aβ.

After initial experiments showed Aβ1‐42 to be most toxic to the CVSMCs, the LIVE/DEAD cell viability assay (Invitrogen, Paisley, UK) was used according to the manufacturer's guidelines to quantify the amount of cell death 24 hours after adding 10 µM Aβ1‐42. For some experiments the CVSMCs were first transfected with NEP siRNA and transfection‐ready NEP cDNA as described above. In other experiments Aβ toxicity was assessed after the addition of thiorphan (2 µM) (Sigma Aldrich, Dorset, UK) or somatostatin (10 µM) (Sigma Aldrich, Dorset, UK) in serum‐free medium 18 hours before incubation with Aβ1‐42. Each experiment was performed independently at least in duplicate and in most cases in triplicate.

Statistical analysis

Data were analyzed by independent‐samples t‐test, one‐ or two‐way ANOVA with Bonferroni post‐testing and Spearman's or Pearson's correlation analysis, as appropriate, with the help of Statistical Package for Social Science software (version 12.0.1 for Windows, SPSS Inc., Chicago, IL, USA). Values of P < 0.05 were considered statistically significant. Most of the data sets were skewed to the right and were normalized by logarithmic transformation for analysis.

RESULTS

Aβ1‐42 is most toxic to CVSMCs

After 24 hours, some cell death was seen in all cultures of CVSMCs (Figure 1) and this appeared to be dose‐related. The amount of cell death was highest on exposure to 10 µM Aβ. At this concentration, cell death was significantly higher after exposure to Aβ1‐42 (∼40%) (P < 0.001) or Aβ1‐40 (Dutch variant) (∼33%) (P < 0.01) than in untreated cells (∼23%); death of CVSMCs incubated with 10 µM Aβ1‐40 (∼29%) and Aβ1‐42(scrambled) (∼28%) did not differ significantly from untreated controls (Figure 1). For further studies, we used 10 µM Aβ1‐42.

Figure 1.

Figure 1

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Aβ toxicity to human cerebrovascular smooth muscle cells (CVSMCs) in vitro. Bar chart showing the percentage cell death at 24 hours caused by adding Aβ in different concentrations to CVSMCs. Error bars indicate the standard error of the mean.

NEP protects CVSMC from Aβ42 peptide‐mediated toxicity

We achieved ∼30% knockdown of both NEP protein concentration (P = 0.007) and enzyme activity (P = 0.047) after transfection of CVSMCs with NEP siRNA compared with untreated cells (Figure 2A). Transfection of CVSMCs with control (scrambled) siRNA had negligible effect on either the concentration or the activity of NEP. Compared with untreated cells, and cells transfected with control siRNA, we observed a ∼20% increase in death of CVSMCs transfected with NEP siRNA, on exposure to 10 µM Aβ1‐42 (P = 0.002) (Figure 2A).

Figure 2.

Figure 2

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NEP protects CVSMCs from Aβ1‐42 cytotoxicity. Reduction in NEP activity was achieved by (A) siRNA knockdown of NEP expression (∼30% reduction in NEP protein, P = 0.007; ∼30% reduction in enzyme activity, P = 0.047) or (B) addition of thiorphan (∼5% reduction in NEP protein, P = 0.046; ∼30% reduction in NEP activity, P = 0.009). Death of CVSMCs on exposure to 10 µM Aβ1‐42 increased ∼20% following siRNA knockdown (P = 0.002) and ∼20% on addition of thiorphan (P = 0.006). Increase in NEP activity was achieved by (C) addition of somatostatin (∼25% increase in NEP protein, P = 0.115; 5% increase in NEP activity, P = 0.730) or (D) transfection of NEP cDNA (∼15% increase in NEP protein, P = 0.046; ∼20% increase in NEP activity, P = 0.026); these manipulations reduced cell death on exposure to 10 µM Aβ1‐42 by ∼15% (P = 0.165) and ∼40% (P = 0.021), respectively. Error bars indicate the standard error of the mean.

Addition of thiorphan [2 µM, a level that specifically inhibits NEP activity (33)] to CVSMCs caused a reduction in NEP activity by ∼30% (P = 0.009) (Figure 2B), whereas NEP protein level was relatively unaffected, being reduced by only ∼5% (P = 0.046) (Figure 2B). Thiorphan‐mediated inhibition of NEP activity was associated with ∼20% increase in CVSMCs cell death on exposure to 10 µM Aβ1‐42 (P = 0.006) (Figure 2B).

Somatostatin is known to induce NEP expression in primary mouse neurons (41). We found that somatostatin (10 µM) increased NEP activity by ∼5% (P = 0.730) and concentration by ∼25% (P = 0.115) in CVSMCs, although the extent of these changes was quite variable and as a result did not reach statistical significance (Figure 2C). Incubation of CVSMCs with somatostatin was associated with reduced (∼15%) Aβ1‐42‐mediated cytotoxicity (P = 0.165) (Figure 2C), although again the extent of cell death was variable and did not reach statistical significance.

Transfection of CVSMCs with full‐length NEP (MMEL1) caused a ∼15% increase in NEP protein (P = 0.046) and ∼20% in NEP activity (P = 0.026) (Figure 2D) compared with measurements in control cells. Transfection with NEP reduced death of CVSMCs exposed to Aβ1‐42 by ∼40% (P = 0.021) (Figure 2D).

Enrichment of SMA and NEP in leptomeningeal and vessel‐enriched preparations

The homogenates of leptomeninges and cerebral cortical vessel‐enriched preparations were considerably enriched in SMA (seven‐ to eightfold, P < 0.0001 and three‐ to fourfold, P < 0.0001, respectively) compared with measurements in crude homogenates of adjacent cortex (Figure 3). The total protein concentration was matched for each analysis.

Figure 3.

Figure 3

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Enrichment of smooth muscle actin (SMA) in leptomeninges and cerebral cortical vessel‐enriched preparations compared with crude brain tissue homogenates. The bar charts show mean SMA levels in the different preparations of meninges or cortex. Compared to measurements in crude brain tissue homogenates of adjacent cerebral cortex, SMA was markedly elevated in leptomeningeal (∼7–8‐fold higher, P < 0.0001) and cerebral cortical vessel‐enriched preparations (∼4–5‐fold higher, P < 0.0001). Error bars indicate the standard error of the mean.

We observed a trend toward reduced SMA in leptomeningeal preparations from AD brains compared with control brains (Figure 3), although in keeping with reports of decreased SMA immunolabeling of cerebral blood vessels in AD 12, 19, 43 the reduction was not statistically significant in our samples. We did not observe any differences in SMA level according to CAA severity.

NEP protein (Figure 4A) and enzyme activity (Figure 4B) in leptomeningeal preparations were compared with the measurements in crude homogenates of adjacent cortex in a small (n = 4) number of cases. Both the concentration (P = 0.03) and activity (P = 0.11) of NEP were higher in the leptomeningeal preparations than in the adjacent cortex, although only the elevation of NEP protein concentration reached significance. In these samples NEP protein and enzyme activity were directly correlated (r = 0.82, P = 0.02), showing that the vascular NEP is enzymatically active (Figure 4C).

Figure 4.

Figure 4

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Increased NEP activity and protein level in leptomeningeal preparations. NEP protein (A) and NEP enzyme activity (B) in leptomeningeal preparations were compared to measurements made on a small number (n = 4) of crude homogenates of adjacent cerebral cortex. The protein level was significantly higher in the leptomeningeal preparations (P = 0.03). There was also a trend toward higher NEP activity (P = 0.11). (C) NEP protein level correlated positively with NEP enzyme activity in the leptomeningeal vessel preparations (r = +0.82, P = 0.02). Error bars indicate standard error of the mean.

Severity and distribution of CAA are related to NEP activity

NEP enzyme activity was lower in the frontal meninges from controls with moderate to severe CAA than from controls with absent or mild CAA (P = 0.005 before adjustment for SMA, P = 0.006 after adjustment) (Figure 5A). Similar although nonsignificant trends were seen for occipital meninges in controls (Figure 5B) and, after SMA adjustment, for frontal and occipital meninges in AD. The findings for NEP concentration were very similar to those for NEP activity in that there was a nonsignificant reduction in NEP concentration in both controls and AD cases with moderate to severe CAA compared with those with absent or mild CAA, in both the frontal and occipital meninges (Figure 5A,B). NEP activity and protein concentration correlated closely across the cohorts (r = +0.682, P < 0.00001). Frontal and occipital leptomeninges did not differ significantly in NEP concentration or activity.

Figure 5.

Figure 5

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Leptomeningeal NEP activity and protein concentration in relation to CAA severity. The bar charts show the measurements of NEP activity and level in the frontal (A) and occipital (B) meninges in relation to CAA severity, in control and AD brains. In the frontal lobe, NEP activity was significantly higher in meninges from controls with absent or mild (0, 1) CAA than in those with moderate to severe CAA (2–4), both before (P = 0.005) and after (P = 0.006) adjustment for SMA concentration. NEP protein concentration was also higher in absent–mild (0, 1) CAA cases but the difference was not significant. (B) In the occipital lobe, NEP activity and protein concentration tended to be higher in controls and AD cases with absent or mild CAA (0, 1) than in those with moderate to severe CAA (2–4). but the differences were not statistically significant. Error bars indicate the standard error of the mean.

In frontal lobe vessel‐enriched homogenates of the cerebral cortex (Figure 6), NEP enzyme activity was lower in both controls and AD with moderate to severe CAA than in those with mild or absent CAA, although not significantly so (Figure 6A). NEP protein concentration did not vary with CAA severity in AD or controls. Neither NEP activity nor NEP protein levels varied with CAA severity in control and AD groups within the occipital lobe (Figure 6B). NEP activity and concentration for combined controls and AD cases, before and after adjustment for SMA, tended to be lower in the occipital than in the frontal cortex (NEP activity, P = 0.075; SMA‐adjusted NEP activity, P = 0.076; NEP concentration, P = 0.146; SMA‐adjusted NEP concentration, P = 0.051), but these differences did not reach statistical significance. NEP activity and protein concentration correlated closely across the cohorts (r = +0.338, P < 0.002).

Figure 6.

Figure 6

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NEP activity and protein concentration in vessel‐enriched cerebral cortex in relation to CAA severity. A. In the frontal lobe, NEP activity and protein concentration were increased in vessel‐enriched preparations from control and AD cases with absent or mild CAA (0, 1) but not significantly so, either for NEP levels or SMA‐adjusted values. B. In the occipital lobe, there were no differences between the cohorts for NEP activity or protein, either before or after SMA adjustment. Error bars indicate the standard error of the mean.

Leptomeningeal Aβ and NEP activity vary with APOE genotype

The concentration of insoluble Aβ in the meninges was related to APOE genotype, being lowest in ε2/ε3 heterozygotes and highest in ε4/ε4 homozygotes (Figure 7). The differences in Aβ concentration with APOE genotype were significant (P < 0.001). Bonferroni post hoc analysis indicated that Aβ load was significantly higher in ε4/ε4 than in ε3/ε3 (P = 0.013) or in ε2/ε3 (P = 0.0002) individuals, and in ε3/ε4 than in ε2/ε3 individuals (P = 0.004).

Figure 7.

Figure 7

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The amount of total insoluble Aβ in leptomeningeal vessels was associated with APOE genotype. The association was highly significant (P < 0.001). Bonferroni post hoc analysis showed the amount of Aβ load to be significantly higher in APOEε4/ε4 than in ε3/ε3 (P = 0.013) or in ε2/ε3 (P = 0.0002) cases. Error bars indicate the standard error of the mean.

SMA‐adjusted NEP activity and concentration within leptomeningeal preparations tended to vary with APOE genotype in the opposite direction to Aβ, being highest in ε2/ε3 heterozygotes and lowest in ε4/ε4 homozygotes (Figure 8). Adjusted NEP activity tended to decline with APOEε4 frequency (r = −0.203, P = 0.073) (Figure 8A). Adjusted NEP protein level varied significantly with APOE genotype (P = 0.029) with Bonferroni post hoc analysis showing significantly increased NEP in ε2/ε3 compared with ε3/ε3 (P = 0.019), ε3/ε4 (P = 0.028) and ε4/ε4 (P = 0.019) cases (Figure 8A).

Figure 8.

Figure 8

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NEP activity in leptomeningeal vessels and vessel‐enriched preparations of cortex was associated with APOE genotype. A. In leptomeninges, SMA‐adjusted NEP activity varied with APOE genotype although this did not quite reach significance (r = −0.203, P = 0.073). SMA‐adjusted NEP protein levels differed significantly with APOE genotype (P = 0.029); Bonferroni post hoc analysis showing significantly higher NEP levels in APOE ε2/ε3 than ε3/ε3 (P = 0.019), ε3/ε4 (P = 0.028) or ε4/ε4 (P = 0.019) cases. B. In vessel‐enriched cortex, NEP enzyme activity did not vary with APOE genotype but the protein level did (P = 0.033), Bonferroni post hoc analysis revealing significantly higher NEP protein in ε2/ε3 than in ε3/ε3 (P = 0.026) cases. Error bars indicate the standard error of the mean.

SMA‐adjusted NEP activity in vessel‐enriched cerebral cortex did not vary with APOE genotype (Figure 8B). Adjusted NEP protein level differed significantly with APOE genotype (P = 0.033) (Figure 8B), a higher level being detected in ε2/ε3 than in ε3/ε3 (P = 0.026).

DISCUSSION

We previously observed reduced immunolabeling of NEP in vessels affected by CAA (32), particularly in association with APOEε4, itself strongly associated with CAA 8, 16, 38. We have now shown that cerebrovascular NEP is enzymatically active; its concentration and activity are reduced in moderate to severe CAA and tend to be lower in the occipital lobe (a site of predilection for CAA) than in the frontal lobe; these reductions are not simply a consequence of loss of CVSMCs; the concentration and activity of NEP in vessels within the leptomeninges are related to APOE genotype, and NEP protects CVSMCs from Aβ1‐42‐mediated toxicity. Our findings point toward an important role for NEP in protecting against CAA and its complications.

Studies of mouse models of AD suggest that NEP is a major Aβ‐degrading enzyme. Ablation of the NEP gene resulted in higher endogenous Aβ load 10, 13, 21. Inactivation of the NEP gene in human APP transgenic mice caused elevation of Aβ1‐40 and Aβ1‐42, whereas over‐expression of NEP reduced Aβ levels 18, 22, 28, 31. NEP also protected murine hippocampal neuronal cells against Aβ peptide toxicity in vitro (11). Although NEP is present in pyramidal neurons, it is also abundant in the cerebral vasculature, within the smooth muscle cells of the tunica media 7, 32. NEP gene silencing in a human APP (J9) mouse model resulted in the development of CAA in a gene‐dosage dependent manner (13). Vessel‐associated NEP immunolabeling in human brain tissue was shown to be reduced in AD and CAA (32), but the biological relevance of this finding has not previously been examined. To minimize potential confounding effects of neuronal NEP in our assays, we have used preparations of brain tissue that consist mostly of blood vessels, from regions susceptible to CAA—lepomeninges and cerebral cortex—as well as primary cultures of CVSMCs. We have thereby been able to measure NEP protein and activity specifically related to blood vessels and CVSMCs.

Our data reveal that NEP is enzymatically active within blood vessels and that NEP enzyme activity within vessel‐enriched preparations is reduced in CAA (even after adjustment for loss of SMA). These findings reflect our earlier observations that NEP immunolabeling is reduced in CAA. Aβ‐mediated degeneration of VSMCs, in both mouse models and in humans, results in the complete replacement of the tunica media with Aβ as the disease progresses to the later stage 56, 62. Loss of NEP in CAA may simply reflect the loss of VSMC. However, we previously showed that in cases with severe CAA, NEP immunolabeling was as markedly reduced in cerebral cortical vessels that were Aβ‐laden as in those that, in the same plane of section, were Aβ‐free, suggesting that NEP loss is not a consequence of Aβ deposition. We have now observed reduced NEP activity in relation to CAA severity, even after adjustment for SMA content, supporting our previous suggestion that NEP levels in the tunica media are globally reduced in individuals with severe CAA independent of Aβ‐mediated loss of muscle cells. Reduced levels of NEP within the vessel wall, in combination with other factors, such as age‐related deficiencies in Aβ drainage along cerebral perivascular pathways 6, 42, 59, are likely to act in concert to predispose individuals to the development of CAA.

Our finding of elevated SMA in leptomeninges and cerebral cortical vessel preparations compared with crude brain tissue homogenates demonstrates good retention and enrichment of small blood vessels. The SMA level in frontal and occipital leptomeninges from AD cases was lower than in controls, as observed by Hullete et al (19), but the difference in our samples did not reach significance. Hullete and colleagues reported that SMA immunolabeling was reduced in blood vessels showing accumulation of Aβ within the tunica media 12, 43. We did not observe a corresponding relationship between SMA content and CAA severity. Tian et al (46) reported that loss of smooth muscle cells correlated with CAA severity in the frontal cortex but not in the occipital cortex, where CAA is generally more pronounced. The authors concluded that the inverse relationship between SMC cell loss and Aβ deposition may become lost as the disease progresses.

APOE genotype strongly influences the risk of developing AD and, particularly in AD patients, the risk of CAA 8, 16, 38 including capillary CAA (45). Although possession of ε4 is associated with increased severity of CAA, ε2 was found to carry an increased risk of cerebral hemorrhage in patients with CAA (36). We have found that insoluble Aβ load within the leptomeninges is strongly influenced by APOE genotype—increasing in a manner that mirrors the risk profile for both CAA severity and AD. An association was reported between APOEε4 and increased cerebrovascular Aβ deposition in gray and white matter arterioles (47), and we previously found that APOEε4 favored cerebrovascular over parenchymal deposition of Aβ(8). The present data support our previous findings that vessel‐associated NEP immunolabeling is reduced in individuals who possess an APOEε4 allele and that reduced vascular NEP is a significant independent predictor of moderate to severe CAA (32). The extent to which APOE influences the severity of CAA through cerebrovascular NEP expression is not known. Our findings suggest that the level of variation in NEP activity in CVSMCs between people of differing APOE genotype may be sufficient to affect the resistance of the CVSMCs to the toxic effects of Aβ. Apolipoprotein E plays an important role in the metabolism of lipids and recycling of cholesterol by neurons (54) and APOE genotype may influence the formation of lipid rafts within which most neuronal membrane‐associated NEP was reported to reside 2, 25; the relevance of these findings to vessel‐associated NEP remains to be determined.

In general, CAA tends to be most severe in the occipital region 3, 55. The explanation for the posterior predominance of CAA is not known. One suggestion is that this distribution reflects the properties of the posterior circulation (58). Reduced vessel‐associated NEP activity in the occipital lobe compared to other regions of cerebral cortex (eg, the frontal lobe, as in the present study) may provide an alternative (or additional) explanation.

Several of the deleterious consequences of CAA, in particular intracerebral hemorrhage, relate to the loss of CVSMCs. Disruption and loss of VSMCs contribute to loss of vasomotor function, impaired vasodilatation and risk of hemorrhage. CAA is a common cause of cerebral hemorrhage, particularly in the elderly (15). The pathogenesis of CVSMC degeneration in CAA has not been explained. The isoform most abundant in vessel walls in established CAA is Aβ1‐40 (15), but we have found that wild‐type Aβ1‐42 is significantly more toxic than Aβ1‐40 to CVSMCs. In similar studies, cell death in cultured human leptomeningeal smooth muscle cells exposed to 25 µM Aβ for 6 days was significantly greater after exposure to Aβ1‐42 than to Aβ1‐40 (53). In contrast, cerebral endothelial cells were more susceptible to Aβ1‐40 (44). We have found that the loss of CVSMCs on exposure to 10 µM Aβ1‐40 (Dutch variant) was similar to that produced by Aβ1‐42, although previous studies found the Dutch variant to be more toxic than Aβ1‐42 to smooth muscle cells 9, 57. The reason for the differences is not entirely clear. It is possible that variations in the extent of aggregation of the synthetic Aβ peptides or in the duration of their exposure to the CVSMCs may account for some of the discrepancies.

Although Aβ1‐40 is the predominant form of Aβ in CAA (15), Aβ1‐42 is probably deposited within the vessel walls at an early stage 23, 24. Alonzo et al (1) showed that cerebrovascular deposits of Aβ grow by the addition of Aβ1‐40 to pre‐existing deposits of Aβ1‐42. Our findings indicate that wild‐type full‐length Aβ1‐42 is substantially more cytotoxic than Aβ1‐40 to CVSMCs. It is possible that the damage caused by the initial accumulation of the more toxic Aβ1‐42 species may serve as a nidus for the subsequent deposition of Aβ1‐40. We have found that the toxicity of Aβ1‐42 to CVSMCs can be moderated by their production of NEP, suggesting that reduced Aβ‐degrading capacity specifically within the tunica media of blood vessels may contribute not only to the development of CAA but also to some of the clinical complications.

Supporting information

Figure S1. The tissue pelleted after homogenization, filtration and sucrose gradient ultracentrifugation of a sample of cerebral cortex consists largely of blood vessels. The bar indicates 250 μm.

Figure S2. Western blot analysis of control and AD brain tissue homogenates shows a major reactive band with an apparent molecular mass of 40 kDa, which is consistent with the predicted molecular weight of smooth muscle actin (SMA). Lane 1 shows molecular weight markers; lanes 3, 5 and 7 are from AD cases; and 2, 4 and 6 from control brains. The membrane was probed with rabbit polyclonal antibody to SMA (1 in 500) (Abcam, Cambridge, UK).

Figure S3. Cerebrovascular smooth muscle cells (CVSMCs) immunolabeled for (A) smooth muscle actin (1:100) and (B) neprilysin (1:20) (Santa Cruz Biotechnology, Tebu Bio, Cambs, UK). The CVSMCs were grown on poly‐L‐lysine (Sciencell, TCS Cellworks, Bucks, UK)‐coated glass coverslips, fixed in 4% paraformaldehyde, washed and incubated with ice‐cold 70% methanol for 5 minutes and 10% donkey serum (Millipore, Watford, UK) for 45 minutes. The primary antibodies were added and left at 4°C overnight. Secondary antibody conjugated to alexa‐fluor 488 (1:500) (Invitrogen, Paisley, UK) was added for 1 hour at room temperature. Images were obtained under a ×60 objective.

Supporting info item

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Supporting info item

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Supporting info item

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ACKNOWLEDGMENTS

This work was supported by the Alzheimer's Research Trust.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. The tissue pelleted after homogenization, filtration and sucrose gradient ultracentrifugation of a sample of cerebral cortex consists largely of blood vessels. The bar indicates 250 μm.

Figure S2. Western blot analysis of control and AD brain tissue homogenates shows a major reactive band with an apparent molecular mass of 40 kDa, which is consistent with the predicted molecular weight of smooth muscle actin (SMA). Lane 1 shows molecular weight markers; lanes 3, 5 and 7 are from AD cases; and 2, 4 and 6 from control brains. The membrane was probed with rabbit polyclonal antibody to SMA (1 in 500) (Abcam, Cambridge, UK).

Figure S3. Cerebrovascular smooth muscle cells (CVSMCs) immunolabeled for (A) smooth muscle actin (1:100) and (B) neprilysin (1:20) (Santa Cruz Biotechnology, Tebu Bio, Cambs, UK). The CVSMCs were grown on poly‐L‐lysine (Sciencell, TCS Cellworks, Bucks, UK)‐coated glass coverslips, fixed in 4% paraformaldehyde, washed and incubated with ice‐cold 70% methanol for 5 minutes and 10% donkey serum (Millipore, Watford, UK) for 45 minutes. The primary antibodies were added and left at 4°C overnight. Secondary antibody conjugated to alexa‐fluor 488 (1:500) (Invitrogen, Paisley, UK) was added for 1 hour at room temperature. Images were obtained under a ×60 objective.

Supporting info item

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Supporting info item

Click here for additional data file. (29.2KB, jpg)

Supporting info item

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