Vascular alterations in PDAPP mice after anti-Aβ immunotherapy: Implications for amyloid-related imaging abnormalities

Alzheimer’s & Dementia 9 (2013) S105–S115

Vascular alterations in PDAPP mice after anti-Ab immunotherapy: Implications for amyloid-related imaging abnormalities Wagner Zago, Sally Schroeter, Teresa Guido, Karen Khan, Peter Seubert, Ted Yednock, Dale Schenk, Keith M. Gregg, Dora Games, Frederique Bard, Gene G. Kinney* Janssen Alzheimer Immunotherapy Research & Development, South San Francisco, CA, USA

Abstract

Background: Clinical studies of b-amyloid (Ab) immunotherapy in Alzheimer’s disease (AD) patients have demonstrated reduction of central Ab plaque by positron emission tomography (PET) imaging and the appearance of amyloid-related imaging abnormalities (ARIA). To better understand the relationship between ARIA and the pathophysiology of AD, we undertook a series of studies in PDAPP mice evaluating vascular alterations in the context of central Ab pathology and after anti-Ab immunotherapy. Methods: We analyzed PDAPP mice treated with either 3 mg/kg/week of 3D6, the murine form of bapineuzumab, or isotype control antibodies for periods ranging from 1 to 36 weeks and evaluated the vascular alterations in the context of Ab pathology and after anti-Ab immunotherapy. The number of mice in each treatment group ranged from 26 to 39 and a total of 345 animals were analyzed. Results: The central vasculature displayed morphological abnormalities associated with vascular Ab deposits. Treatment with 3D6 antibody induced clearance of vascular Ab that was spatially and temporally associated with a transient increase in microhemorrhage and in capillary Ab deposition. Microhemorrhage resolved over a time period that was associated with a recovery of vascular morphology and a decrease in capillary Ab accumulation. Conclusions: These data suggest that vascular leakage events, such as microhemorrhage, may be related to the removal of vascular Ab. With continued treatment, this initial susceptibility period is followed by restoration of vascular morphology and reduced vulnerability to further vascular leakage events. The data collectively suggested a vascular amyloid clearance model of ARIA, which accounts for the currently known risk factors for the incidence of ARIA in clinical studies. Ó 2013 The Alzheimer’s Association. All rights reserved.

Keywords:

Vascular amyloid; Immunotherapy; Microhemorrhage; ARIA

1. Introduction b-amyloid (Ab) plays a central role in the neuropathology and genetics of Alzheimer’s disease (AD) [1]. Reduction of Ab production and induction of its clearance have been advanced as putative therapeutic approaches for the treatment All authors, except Karen Khan, were/are employees of Janssen Alzheimer Immunotherapy R&D, LLC. Wagner Zago, Sally Schroeter, Teresa Guido, Peter Seubert, Ted Yednock, Dale Schenk, Keith Gregg, Dora Games, Frederique Bard, and Gene Kinney were/are employees and/or shareholders of Elan Pharmaceuticals. Karen Khan was an employee of Janssen Alzheimer Immunotherapy R&D at the time of study conduct and was/is a shareholder of Elan Pharmaceuticals. This study was sponsored by Janssen Alzheimer Immunotherapy R&D, LLC, and Pfizer, Inc. *Corresponding author. Tel.: 1650-615-2110; Fax: 1650-837-8560. E-mail address: [email protected]

of AD [2,3]. Many studies have demonstrated that immunotherapy targeting brain Ab in human APP (hAPP) expressing transgenic mice reduces Ab pathology [4,5]. These studies further supported the advancement of active and passive anti-Ab immunotherapy to clinical evaluation for the treatment of AD. Bapineuzumab is a humanized monoclonal antibody that specifically targets the N-terminus of the Ab peptide. Treatment of AD subjects with bapineuzumab for 78 weeks significantly reduced fibrillar brain Ab relative to placebo-treated controls, as demonstrated by 11C-Pittsburgh compound B (11C-PIB) positron emission tomography (PET) imaging in Phase 2 and 3 studies [6,7]. However, treatment with bapineuzumab has also been associated with the appearance of sulcal and parenchymal hyperintensities, initially characterized as vasogenic edema

1552-5260/$ - see front matter Ó 2013 The Alzheimer’s Association. All rights reserved. http://dx.doi.org/10.1016/j.jalz.2012.11.010

S106

W. Zago et al. / Alzheimer’s & Dementia 9 (2013) S105–S115

(VE), on magnetic resonance imaging (MRI) [8,9]. More recent studies have reported similar MRI abnormalities in untreated AD and in cerebral amyloid angiopathy (CAA) patients [10,11] as well as after treatment with the notch-sparing g secretase inhibitor BMS-708163 [12] or the anti-Ab antibody gantenerumab [13]. The Alzheimer’s Association Research Roundtable recently reviewed these clinical imaging abnormalities and concluded that the abnormalities, previously characterized as VE and microhemorrhages, represent a spectrum of events that may share common underlying pathophysiological mechanisms and subsequently adopted the more accurate nomenclature of amyloid-related imaging abnormalities (ARIA) [15]. An increased risk of ARIA associated with apolipoprotein ε4 (APOE ε4) genotype suggested that vascular amyloid burden may be a common feature associated with ARIA [14]. With bapineuzumab and gantenerumab, the risk of ARIA increased with the dose of the antibodies [9,13,14], suggesting a relationship between the effectiveness of amyloid clearance and the imaging abnormality. Finally, it has been shown that treatment-related ARIA are often transient and that the risk of ARIA decreases with an increasing number of bapineuzumab infusions [9]. The transient nature of ARIA suggests a biological process that resolves or compensates after prolonged treatment. Bapineuzumab was humanized from the murine anti-Ab antibody 3D6 [8,16,17]. Accordingly, the present report describes preclinical findings after chronic 3D6 treatment of hAPP mice that collectively provide insight into the mechanisms underlying the occurrence of microhemorrhage events. The relationship to ARIA in AD patients, particularly those expected to have increased vascular Ab (VAb) burden (APOE ε4 genotype) is further discussed. On the basis of clinical and preclinical observations, we propose a model of ARIA involving vascular clearance of Ab.

Fig. 1. Study design. PDAPP mice were immunized with 3 mg/kg/week of 3D6 or IgG control (TY11-15 or IB4) for 1 or 7 weeks (starting at 18 months of age) or 12–36 weeks (starting at 12 months of age).

2.2. Brain tissue preparation for histochemistry Animals were deeply anesthetized with isofluorane and perfused with saline intracardially. One hemisphere from each brain was immersion-fixed for 48 hours in 4% paraformaldehyde at 4
C and sectioned coronally at 40 mm on a vibratingblade microtome or embedded into blocks of gelatin and sectioned on a sliding microtome (NeuroScience Associates, Knoxville, TN). The sections were stored in antifreeze solution (30% glycerol/30% ethylene glycol in 40 mM Na2HPO4, pH 7.4) at 220
C before immunostaining. Four to six sections, spanning the rostral hippocampus at 240-mm intervals, were selected from each brain for analysis. Sections were stained and analyzed by investigators blinded to the treatment status. 2.3. Ab deposits and microhemorrhage co-labeling histochemical procedure Ab deposits were labeled with biotinylated antibody 3D6 in 1% horse serum in phosphate buffered saline (PBS) overnight at 4
C. The floating sections were then reacted with an avidin-biotinylated horseradish peroxidase complex and developed using 3,3-diaminobenzidene. Sections were mounted on slides and co-stained with a Perls iron reaction [20] modified by incubation at 37
C to intensify the hemosiderin reaction product.

2. Methods

2.4. Leptomeningeal VAb quantification

2.1. Study design

3D6-immunoreactive blood vessels were assessed by counting the number of all amyloid-containing vessels in the sections (n 5 4–6/animal) as described previously [18]. In brief, vessels were counted if they contained any amount of amyloid and thus included partially cleared and noncleared vessels. The counts were then analyzed using a receiver operating characteristic (ROC) curve to determine cutoffs for classification in the two resulting categories of leptomeningeal VAb: “none to little VAb” (3 amyloidpositive vessels per section per animal) or “moderate VAb” (.3 amyloid-positive vessels per section per animal). Results were then represented as the percentage of animals showing Ab deposition in cortical leptomeningeal vessels.

To investigate the vascular changes associated with antiAb immunotherapy, we performed a time course analysis in plaque-bearing PDAPP mice immunized with 3D6 for periods ranging from 1 to 36 weeks. To accommodate the course of disease progression and the age span of mice, we designed two independent experiments: one starting at 12 months of age (with 12–36 weeks of treatment) and another starting at 18 months of age (with 1–7 weeks of treatment) (Fig. 1). Mice were injected intraperitoneally once weekly at 3 mg/kg after a loading dose of 7.5 mg/kg with antibody 3D6 (IgG2a) or control isotype-matched antibodies (IB4 or TY11-15). The number of mice in each treatment group ranged from 26 to 39 per time point. The dose of 3D6 antibody used in the current study was previously reported to promote clearance of parenchymal plaques and VAb [18,19].

2.5. Capillary Ab quantification The prevalence of 3D6-immunoreactive capillaries was assessed similarly as described for VAb. Six sections per brain

W. Zago et al. / Alzheimer’s & Dementia 9 (2013) S105–S115

were evaluated, and animals were categorized for the presence and severity of capillary Ab deposits. Capillary Ab deposits were restricted to four brain regions: the medial cortex, the hippocampus, the fimbria, and the corpus callosum. Ratings were assigned to each of these regions separately for each brain. 2.6. Microhemorrhage quantification Each animal was scored on a two-level scale for the presence, amount, location, and intensity of hemosiderin staining across four to six brain sections. A score of “none-little” indicated absence or presence of small puncta in less than three brain sections and a score of “moderate” was assigned to brains showing contiguous accumulations of hemosiderin and greater staining intensity in most of the sections. These ratings were designed to reflect the range of hemosiderin-positive staining observed in the present animal studies and therefore do not represent or translate to ratings of clinical hemorrhagic disorders. 2.7. Immunostaining and analysis of vascular elements To understand the nature of vascular degeneration caused by vascular amyloid deposition and assess the effect of immunotherapy, we performed morphological analyses of two major vascular components within leptomeningeal vessels, the smooth muscle cell (SMC) layer and the extracellular matrix (ECM). Representative animals (n 5 6–8/group) of each treatment group were selected based on their proximity to median values of vascular amyloid deposition. Brain sections were immunolabeled in free-floating mode or mounted on glass slides. Brain sections were suspended in 10 mM citrate buffer (pH 6.0, Invitrogen, USA) and heated at 95 6 5
C for 10 minutes and then treated with the proteolytic enzyme collagenase type IV (0.1 mg/mL; Invitrogen) during the incubation of primary antibodies overnight. Immunostaining for Ab (mouse monoclonal, 3D6 conjugated to Alexa Fluor 647), smooth muscle a-actin (mouse monoclonal conjugated to CY3, Sigma, St. Louis, MO), ferritin (goat polyclonal, Santa Cruz, Santa Cruz, CA), and the ECM component collagen IV (rabbit polyclonal, Millipore, Billerica, MA) was performed overnight at room temperature. Nonspecific staining was assessed by substituting primary antibodies with immunoglobulin G (IgG, Vector Laboratories, Burlingame, CA) from the same host. Sections were mounted with ProlongGold (Invitrogen) and analyzed using a BX61 Olympus or SPE Leica confocal microscope. Series of 30 to 100 optical z-stacks at 0.3-mm intervals were acquired and postprocessed by deconvolution. To assess the levels of VAb and the elements composing the vascular wall, we developed a software algorithm that determined the wall thicknesses and the relative distribution of amyloid within the vascular layers. Lines were originated from the centroid of crosscut sections to the abluminal limits of vessels at 2
intervals (180 lines total). For each line, a map of pixel intensity values was generated and, based on fixed threshold values, the area occupied by each one of the markers was determined (amyloid, collagen IV, and ferritin).

S107

2.8. Aquaporin-4 levels in capillaries To investigate microvascular changes associated with newly formed capillary Ab deposits, we assessed the levels of the water channel aquaporin-4 (AQP4) by immunohistochemistry in PDAPP mice treated with 3D6. Immunostaining was performed for Ab (mouse monoclonal 3D6 conjugated to Alexa Fluor 647), glial fibrillary acidic protein (GFAP; mouse monoclonal conjugated to CY3, Sigma), AQP4 (rabbit polyclonal, Millipore), and the ECM component laminin (chicken polyclonal, GenWay, San Diego, CA). Sections were mounted with ProlongGold and analyzed using a BX61 Olympus microscope or Leica SPE confocal microscope. Series of 10 to 20 optical z-stacks at 0.1-mm intervals were acquired and postprocessed by deconvolution. 2.9. Transmission electron microscopy The vibratome brain sections were cryofixed using a RMC-HPM 010 high-pressure freezing machine. After fixation, sections were cryosubstituted in 100% acetone containing 0.1% uranyl acetate and embedded in Lowicryl resin (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections of the areas of interest were cut and placed on Formvarcoated nickel grids (Electron Microscopy Sciences). The sections were blocked in 5% normal goat serum/5% bovine serum albumin (BSA)/0.1% cold water fish skin gelatin in PBS for 30 minutes, stained with anti-aquaporin antibody (rabbit polycolonal, Millipore) in PBS containing 0.1% acetylated BSA (Aurion, Wageningen, The Netherlands) at 1:200, and immunogold-labeled with 10-nm gold particleconjugated goat anti-rabbit (Aurion) diluted in PBS containing 0.1% acetylated BSA at 1:10. Sections were viewed on a Hitachi H7500 transmission electron microscope. 2.10. Statistical methods Prevalence of leptomeningeal VAb, microhemorrhages, and capillary Ab are presented as frequencies and percentages and analyzed using Fisher’s exact test. For parenchymal Ab burden, differences among groups were examined by Mann-Whitney tests. Variance in vessel wall thickness was analyzed using one-way analysis of variance with the Dunnett multiple comparison procedure for comparing wild-type and PDAPP-3D6 to control for each time point. A criterion for statistical confidence of P , .05 was adopted. 3. Results 3.1. Dynamics of parenchymal Ab and VAb clearance by immunotherapy with 3D6 We performed an extended time course analysis of vascular and parenchymal amyloid levels in plaque-bearing PDAPP mice immunized with 3D6 for periods ranging from 1 to 36 weeks. At the start of treatment, at either 12 or 18 months of age, the prevalence of leptomeningeal VAb was 25% and 50%, respectively. After passive

S108

W. Zago et al. / Alzheimer’s & Dementia 9 (2013) S105–S115

Table 1 Occurrence of parenchymal and vascular (leptomeningeal and capillary) Ab deposits in PDAPP mice treated with 3 mg/kg/week 3D6 for periods of 1–36 weeks Treatment Parenchymal Ab period burden (% IgG (weeks) control; mean 6 SD) 1 7 12 24 36

101 6 91% (n 5 39) 81 6 68% (n 5 35) 26 6 35% (n 5 41)y 18 6 18% (n 5 35)y 7 6 9% (n 5 33)y

Prevalence of Prevalence of capillary Ab leptomeningeal Ab (% of 3D6-treated (% IgG control) animals) 81% (n 5 39) 78% (n 5 34) 14% (n 5 41)y 15% (n 5 37)* 8% (n 5 34)y

0% (n 5 39) 3% (n 5 32) 7% (n 5 41) 62% (n 5 37)y 35% (n 5 34)yz

NOTE. Parenchymal Ab values were normalized to the median in the IgG group. Capillary Ab deposits were absent in the control IgG group (n 5 31– 41). P values for parenchymal Ab are based on a Mann-Whitney test on the un-normalized values. P values for prevalence of leptomeningeal Ab and capillary Ab comparing 3D6 with IgG control are based on Fisher’s exacts tests with a Bonferroni multiple comparison procedure applied across the 10 comparisons. The P value for prevalence of capillary Ab comparing 3D6 at 24 at 36 weeks is based on Fisher’s exacts test. *P , .01 compared with control IgG. y P , .001 compared with control IgG. z P , .05 compared with 24 weeks of treatment.

immunotherapy with 3D6, parenchymal Ab and VAb deposits were cleared in a time-dependent manner (Table 1). Of note, parenchymal and vascular endpoints appeared to follow a similar time course of clearance, with 70% to 80% removal observed at 12 weeks (Table 1). Although the prevalence of vascular deposits was not significantly reduced after 7 weeks of treatment, we observed examples of “patchy” Ab morphology suggesting partial clearance at that time point (Fig. 2B). 3.2. Appearance and resolution of microhemorrhage after immunotherapy with 3D6 Ab immunotherapy in mouse models has been associated with increased incidence of vascular microhemorrhages

depicted by microscopic bleeds detected by hemosiderin staining [18,21]. To determine the time course of microhemorrhage in relation to VAb clearance, we investigated the presence of hemosiderin deposits colabeled with VAb in PDAPP mice treated from 1 to 36 weeks with 3D6. We detected hemosiderin deposits in leptomeningeal vessels as early as 7 weeks after treatment with 3D6 (P , .0001, compared with IgG control) when it affected approximately 37% of animals. These deposits were invariably present within the boundaries of the leptomeningeal vessel walls, and no signal was detected in the parenchyma, hence the use of the term “microhemorrhage”. The prevalence of microhemorrhages did not appear to increase further in animals treated for 12 or 24 weeks (27% and 38% of animals, respectively) (Fig. 2A), a time period in which VAb clearance was maintained near maximal levels. During the initial stages of Ab clearance (7–12 weeks), hemosiderin deposits were found in leptomeningeal vessels and in close proximity to eroded Ab deposits (Fig. 2B), suggesting that microhemorrhages occurred at sites of active clearance by the antibody. With prolonged treatment, hemosiderin granules were often found inside of macrophages (Fig. 2E), as previously reported [22]. After 3D6 treatment for 36 weeks, there was a reduction in the occurrence of microhemorrhages to levels that were not statistically different from those of control IgG-treated animals (P . .05). 3.3. Evidence of restoration of vascular structure after Ab clearance Independent of anti-amyloid therapy, structural disruption of vascular smooth muscle by amyloid can lead to weakness and rupture of the vessel wall, resulting in microhemorrhagic events and dysregulation of the neurovascular unit [23]. In the PDAPP mice used in the present study, VAb was almost invariably found around and within the SMC layers that compose the tunica media, often associated

Fig. 2. Immunotherapy with antibody 3D6 transiently increases the occurrence of microhemorrhages in leptomeningeal vessels of PDAPP mice. (A) Prevalence of microhemorrhages in PDAPP mice (% of animals) measured by categorizing animals in “none-little” or “moderate”. At 36 weeks of immunotherapy, the occurrence of microhemorrhages in 3D6-treated mice returns to levels not different from control IgG-treated animals (P . .05). (B, C) Example of a leptomeningeal vessel with partial clearance of amyloid deposits (brown) and appearance of hemosiderin (blue) at 7 weeks of treatment. (C) Higher magnification. (D, E) Representative images of microhemorrhage in leptomeningeal vessel at 24 weeks of treatment. (E) Hemosiderin deposits in phagocytic cells. n 5 26–39 animals/group. *P , .05 compared with control IgG-treated animals by Fisher’s exact test.

W. Zago et al. / Alzheimer’s & Dementia 9 (2013) S105–S115

with disorganization and degeneration of SMC and ECM (Fig. 3B). By comparing the topological maps of vessels from PDAPP mice with those of wild-type mice, we observed that many vessels showed increased wall thickness, suggestive of hypertrophy and/or hyperplasia (Fig. 3B and C), as well as areas of hypotrophy/atrophy. It is important to note that the variance in wall thickness was not due to the intercalation of Ab between SMCs because that space was omitted from the measurements.

S109

The hypertrophic and degenerative regions of vessels were invariably adjacent to amyloid deposits (Fig. 3C, red dots) and appeared to correlate with the amount of amyloid (Fig. 3C, size of dots). To perform analyses that encompassed the two phenotypes (i.e., hypertrophy and atrophy), we determined values of variance in SMC and ECM wall thicknesses along the circumference of the vessel (Fig. 4). We observed that the variance of muscle and elastic layers for PDAPP mice were significantly increased in relation to wild-type mice (P , .001 and P , .05, respectively). The increase in variance appeared to be a direct effect of amyloid deposition because PDAPP vessels lacking Ab deposits did not manifest the phenotypic changes (P . .05). Next, we examined the effects of immunotherapy on vascular morphology in mice treated with 3D6. Immunization with 3D6 promoted a time-dependent reversal in the amyloid-related vascular phenotype, reducing the variance in wall thickness for ECM and SMC to levels found in wild-type vessels (Figs. 3C and 4). Although we observed a trend toward increased variance in ECM thickness after 1 and 7 weeks of treatment, it was not significant (P . .05 comparing with IgG control). To more directly determine if the subpopulation of vessels that previously contained amyloid deposits fully recovered their morphology, we performed measures in the subpopulation of vessels that contained ferritin deposits, a core and persistent component of hemosiderin. Because ferritin was not found in wild-type mice and only found in vessels from transgenic mice co-labeled with altered or reduced Ab morphology, the presence of ferritin is an indicator that at some point in the past those vessels demonstrated microhemorrhage, 200

Variance of thickness (%IgG control)

Smooth Muscle ( -actin) 150

Elastic Layer (Collagen IV)

100

*

** 50

**

** ***

***

36 (F+)

wild type

*** 0 1

Fig. 3. Disruption of leptomeningeal vascular structure by Ab deposits in PDAPP mice and resolution after immunotherapy with 3D6. (A, B) Top: Representative images reconstructed from leptomeningeal vessels containing or not containing Ab deposits. Note the degeneration of smooth-muscle layer (a-actin, green) in the vicinity of amyloid deposits (red). Bottom: Individual vascular plots of vessels in panel A and B showing increased variability in smooth-muscle thickness in PDAPP vessels. (C) Pooled vascular plots from wild-type or PDAPP mice treated with either 3D6 or IgG control (TY11-15) for 36 weeks. Note the resolution of phenotypic changes to wild-type levels in 3D6 treated PDAPP mice. n 5 43–70 vessels/treatment group, 6–8 animals/group. Red symbols represent regions of the vessels adjacent to VAb deposits and the size is proportional to the extent of amyloid deposition.

7

12

36

Treatment Period (Weeks)

Fig. 4. Immunotherapy with 3D6 normalizes the structural disruption of vascular components in a time-dependent manner. Variance in vascular smooth-muscle (black) and elastic layer (gray) in the midline leptomeningeal vessels of wild-type or PDAPP mice treated with 3D6 for 1–36 weeks. “36 (F1)” represents a subpopulation of vessels containing ferritin deposits. Data were normalized to the IgG control group (TY11-15 or IB4) and are represented as mean 6 SEM of coefficient of variance in thickness per vessel. n 5 43–70 vessels/group, 6–8 animals/group. *P , .05, **P , .01, and ***P , .0001 compared with control IgG-treated animals using one-way analysis of variance.

S110

W. Zago et al. / Alzheimer’s & Dementia 9 (2013) S105–S115

presumably in association with treatment related to the clearance of VAb. It is interesting to note that after 9 months of treatment, ferritin-positive vessels were found to have variance values not different than wild-type controls (P . .05, Fig. 4). These data collectively suggest that the clearance of vascular amyloid deposits after immunotherapy with 3D6 led to a restoration of vessel wall integrity to wildtype conditions. 3.4. Capillary Ab Previous reports indicated that passive and active anti-Ab immunotherapy increase vascular amyloid on capillary structures [21,24]. Consistent with those reports, we found that there was an increase in the level of capillary Ab accumulation after immunotherapy with 3D6, mainly localized in the medial cortex and to a lesser degree in the corpus callosum, hippocampus, and fimbria. Capillary accumulation was increased after 12 to 24 weeks of immunotherapy and decreased after 36 weeks of treatment (Table 1). Of note, to a lesser extent, spontaneous accumulation of capillary Ab was observed in untreated PDAPP mice at 18 months of age affecting the same four brain regions. 3.5. Capillary Ab deposits cause downregulation and redistribution of AQP4 channels AQP4 is a bidirectional water channel that facilitates reabsorption of excess fluid during conditions of brain edema [25]. After 3D6 treatment, we found that the newly formed capillary Ab deposits promoted focal loss of perivascular AQP4 (Fig. 5A and B), as previously described in nonimmunized hAPP mice [26,27]. The specificity of this focal decrease in AQP4 immunostaining was supported by a lack of change in reactivity to other capillary vascular elements (not shown). In addition, AQP4 levels in capillaries that crossed the dense cores of parenchymal Ab plaques were not affected (Fig. 5C and D). Capillary AQP4 loss was also observed in aged (18 months) PDAPP animals that spontaneously developed capillary Ab deposits, further supporting that the observed phenomenon was due to the deposition of Ab on capillaries and was not restricted to the context of immunotherapy. To investigate whether AQP4 loss was associated with changes in astrocytic endfeet, we examined the distribution of the astrocyte marker GFAP in areas of capillary deposits. We found that Ab intercalated within the vascular unit and spatially disrupted the astrocyte endfeet encircling endothelial cells (Fig. 5E). Ultrastructural analysis confirmed that capillary Ab promoted structural disorganization of the astrocyte endfeet and disrupted their normally tight contact with endothelial cells (Fig. 5F and G). Ultrastructural localization of AQP4 in astrocytes by immunogold labeling confirmed that capillary Ab displaced membrane structures expressing AQP4 away from contact with capillaries (Fig. 5G). It is well established that Ab localizes within small blood vessels (arterioles and

Fig. 5. Changes in pericapillary structure associated with VAb deposits in PDAPP mice. (A, B) Capillaries with Ab deposits (white) present focal loss of the water channel AQP4 (green, arrowheads) Scale bar 5 100 mm. (C, D) Normal expression of AQP4 in capillaries associated with Ab plaques (white). Scale bar 5 200 mm. (E) Ab deposits (white) intercalate in the astrocyte endfeet (GFAP, red) encircling endothelial cells and promote focal AQP4 loss (green). Scale bar 5 20 mm. (F, G) Composite electron micrograph depicting disruption of the anatomical relationship between perivascular astrocyte endfeet and endothelial cell. Note the gold particles from AQP4 labeling (G, arrowheads) showing ectopic expression of the channel in contact with Ab deposits. Scale bar 5 0.2 mm. (H) Vessels from AD patient- and age-matched control subject showing downregulation of AQP4 levels associated with capillary Ab deposition and ectopic expression of AQP4 on astrocyte processes. Scale bar 5 20 mm.

W. Zago et al. / Alzheimer’s & Dementia 9 (2013) S105–S115

capillaries) in AD patients [28]. Thus, we further evaluated whether the changes in AQP4 observed in our mouse studies also occurred in AD brain. Capillary Ab deposits were found in cortical areas in all of the six AD brains that we examined and were invariably associated with loss of focal AQP4 immunoreactivity. It is interesting to note that in cortical areas with a high density of capillary Ab deposition, AQP4 was observed in the soma and processes of astrocytes (Fig. 5H), suggesting a redistribution of the water channel from the endfeet to the soma of these glial cells. 4. Discussion ARIA was first noted in clinical studies evaluating the effect of bapineuzumab [8,9], a humanized anti-Ab monoclonal antibody that reduces central fibrillar Ab in mouse models and AD patients [6]. After these initial observations, ARIA has been noted in untreated AD and CAA patients [10,11] and after treatment with the anti-Ab antibody gantenerumab as well as the notch-sparing g-secretase inhibitor BMS-708163 [9,12–15]. Several associated observations have been noted in AD studies [9,14,15]. These observations include a higher incidence of ARIA amongst APOE ε4 carriers, a higher incidence of ARIA associated with higher doses of the anti-Ab antibodies [13], a lower cumulative risk of developing ARIA with increased number of bapineuzumab infusions, the transient nature of ARIA due to VE and/or sulcal effusions (i.e., ARIA with edema [ARIA-E]) in most cases, and the transient nature of ARIA-E in a few AD patients in which bapineuzumab treatment was continued throughout ARIA-E events. Further, in one case, a symptomatic patient was reported to respond to intravenous steroid treatment [15]. Collectively, these risk factors and associated clinical observations suggested that ARIA may be related to vascular amyloid burden in the context of the disease and vascular amyloid clearance in the context of treatment [14]. Although no animal models have yet been described that completely recapitulate the imaging aspects of ARIA, several reports have evaluated the role of immunotherapy on amyloid deposition and clearance in the cerebral vasculature. The effect of immunotherapy on VAb and occurrence of microhemorrhage has been variable in different transgenic mouse models [29]. Vascular microhemorrhage has been reported in several preclinical studies using antibodies that are targeted to either N- or C-terminal epitopes, which also mediate clearance of fibrillar Ab [18,21,30]. By contrast, the mid-domain antibody 266, which less efficiently recognizes and clears deposited Ab [19], does not appear to cause microhemorrhage in animal models [18,30]. To the extent that microhemorrhage may represent transvascular leakage events, we reasoned that a study of the time course and underlying cause of these events could provide a common biological understanding relevant to clinical ARIA. Most studies that have evaluated the effect of immunotherapy on VAb have done so at a single time point that varies

S111

between 6 weeks and 6 months of treatment and have not typically distinguished between deposits in large vessels versus capillaries. Also, the present study is the first to examine co-labeled microhemorrhage and VAb morphology. Thus, this more thorough understanding of the relationship between VAb clearance and/or capillary Ab accumulation and increased microhemorrhage was of value. An extended passive immunotherapy study with 3D6, the murine antibody form of bapineuzumab, in PDAPP transgenic mice and with analysis at several time points (12, 24, and 36 weeks) allowed for a characterization of the spatiotemporal relationship of VAb clearance and the appearance of microhemorrhages. Much of the VAb clearance occurred within the first 12 weeks of treatment (86% reduction). By 36 weeks, near complete clearance of parenchymal and VAb was noted as compared with the control group. Thus, passive immunotherapy with 3D6 induced parenchymal and vascular amyloid clearance in PDAPP mice with a similar time course. The clearance of VAb by 3D6 treatment was first accompanied by an increase in microhemorrhages as measured by the detection of focal deposits of hemosiderin. The close proximity of those hemosiderin deposits with eroded VAb deposits suggests that leptomeningeal vessels containing vascular amyloid are predisposed to microhemorrhage during the initial phases of Ab clearance by immunotherapy. The increased occurrence of microhemorrhages by 3D6 appeared to be a transient phenomenon because, by 36 weeks of treatment, microhemorrhages returned to baseline level. A persistence of hemosiderin within tissues is well established [31]; therefore, it is likely that a decrease in new microhemorrhage events occurred earlier than 36 weeks. Nonetheless, the reduction of microhemorrhages observed at 36 weeks in these studies contrasts with the general belief that microhemorrhage detected in human subjects by techniques such as MRI using gradient recalled echo (GRE) sequences are irreversible. These findings may indicate different microhemorrhage characteristics such as size and/or a differential sensitivity between the histopathological techniques used in the present preclinical studies versus imaging methods used in the clinical setting. To fully understand these differences, it may be necessary to develop approaches that can distinguish between new microhemorrhage events and the residual effects of past events with unknown persistence. An additional difference between the current studies in PDAPP mice and AD was the relative lack of microhemorrhage in nonimmunized PDAPP mice. By contrast, brain microhemorrhage is observed in untreated AD (16%–32%) (for review see [32] and [33]). That said, the anatomical distribution of spontaneous microhemorrhage in AD is commonly adjacent to VAb deposits, which is consistent with our findings in PDAPP mice after immunotherapy and may suggest the involvement of CAA pathology and associated disruption of vascular architecture [23]. It is interesting to note that APOE ε4 carriers are at increased risk for CAA related damage of vascular structure and

S112

W. Zago et al. / Alzheimer’s & Dementia 9 (2013) S105–S115

have been reported to be at increased risk for amyloid deposition [34,35]. Given the inherent vascular instability caused by Ab, it is not surprising that direct removal of amyloid from the vascular wall may uncover the underlying vascular pathology associated with CAA and contribute to the development of transvascular leakage events such as vasogenic edema or microhemorrhage (i.e., ARIA-E or ARIA with microhemorrhage [ARIA-H]). Quantification of vascular integrity in PDAPP mice demonstrated that changes in the cerebral vascular wall were invariably associated with the presence of VAb deposits. These changes included degeneration (decreased thickness) and hyperplasia/hypertrophy (increased thickness) of SMC and ECM, and both findings were often observed in the same vessel. Such changes were not present in wild-type animals or PDAPP vessels lacking amyloid. The extreme degrees of thickening and thinning of the smooth muscle resulted in a widely variable vascular phenotype in untreated PDAPP mice. Passive immunotherapy with 3D6 restored the pattern of vascular SMC and ECM thicknesses and reduced the phenotypic variability in a time-dependent manner, reaching control levels (wild type) at 36 weeks of treatment. These data suggest that vascular amyloid clearance allowed for the recovery of meningeal vessels from amyloid-induced structural damage. It is noteworthy that this recovery was associated with a decrease in microhemorrhages, which further suggests that mechanisms of repair may be triggered by VAb removal, ultimately leading to recovery from vascular dysfunction. CAA in AD occurs in the walls of leptomeningeal arteries in the subarachnoid space and in the walls of arteries and capillaries in the brain [33]. Vascular amyloid deposition in the PDAPP transgenic mouse is mainly associated with leptomeningeal vessels [18]. Capillary Ab deposits are infrequently observed in nontreated transgenic mice, but, as demonstrated in the present report, newly formed capillary Ab deposits are often seen in immunized mice. Over the time course studied in the present report, an increase in capillary Ab was observed within the first 24 weeks of treatment. By 36 weeks of treatment, there was a decrease in capillary Ab when compared with the 24-week time point (Table 1). Thus, the increase in capillary Ab that accompanied amyloid clearance was transient. A parsimonious explanation for the simultaneous increase in capillary Ab and persistent decrease in leptomeningeal VAb after 3D6 treatment is that displacement of parenchymal Ab to the perivascular clearance pathway occurs after treatment. With continued treatment, as VAb clearance is perpetuated and morphology is restored to wild-type conditions, recovery of the capacity of this clearance pathway could lead to decreased accumulation of capillary Ab. Further research is needed to evaluate the relationship between perivascular clearance of Ab and the effects of anti-Ab immunotherapy on central pathological endpoints. To further assess the relevance of cerebrovascular amyloid-associated changes at the capillary level to ARIA, we evaluated the expression of the water channel AQP4 in

areas of newly formed capillary Ab deposits in 3D6-treated animals. Capillary Ab deposits were consistently associated with focal loss of AQP4. Of note, the focal decrease in AQP4 was due to the deposition of amyloid on capillaries and did not require formation of immune-complexes after immunotherapy in that similar AQP4 loss was observed in aged (18 month old) nonimmunized PDAPP animals that spontaneously developed low levels of capillary Ab. Ultrastructural analyses confirmed that capillary Ab promoted structural disorganization of the perivascular astrocytic endfeet and disruption of their relationship with endothelial cells. Immunogold labeling of AQP4 in brain astrocytes confirmed displacement of the water channel on membrane structures at sites of capillaries with Ab (see also [27]). Analysis of AD and non-AD brain sections confirmed that the microvascular changes observed in PDAPP mice also extend to AD. All of the AD sections analyzed showed VAb deposits in arterial and capillary structures, consistently surrounding the perimeter of the vessels. Consistent with the findings in PDAPP mice, capillary Ab deposits in AD brain sections were invariably associated with a loss of localized AQP4 immunohistochemical staining. It is interesting to note that in areas with a high number of affected vessels, aberrant expression of AQP4 was observed in the soma of astrocytes, suggesting a redistribution of AQP4 from the endfeet to the soma of these astrocytes. Because these brain samples were obtained from AD patients that had not been treated with anti-amyloid therapy, the results may be more aligned with our observations in aged PDAPP mice (18 months) in which spontaneous capillary Ab deposition was noted, presumably because of the advanced disease state. Nonetheless, these data highlight important microvascular changes associated with the deposition of Ab in capillaries, which is promoted after anti-Ab immunotherapy in PDAPP mice. AQP4 channels are critical for the maintenance of water balance in the brain, which suggests a potential role for AQP4 redistribution in the detection of ARIA-E. Because ARIA-E is detected as an MRI abnormality using fluidattenuated inversion recovery (FLAIR) sequences [15], any biological process that results in an increased residence time of parenchymal fluid could lead to an increased incidence of detection. On the basis of the currently reported clinical experience with ARIA and our studies using PDAPP mice, we propose a vascular amyloid clearance model of ARIA related to antiAb immunotherapy (Fig. 6). Under normal conditions Ab is expected to clear from the brain via multiple pathways, including the perivascular drainage pathway [36,37]. With aging [38], increased risk of CAA (e.g., APOE ε4 genotype), and other potentially inflammatory vascular components [39], the capacity of this pathway becomes limited and Ab accumulates in arterial walls. This deposition induces damage in the wall integrity with disruption of SMC. As this damage increases, spontaneous transvascular leakage events may occur at a low incidence [10,11]. As VAb is cleared after immunotherapy, the underlying impaired vasculature

W. Zago et al. / Alzheimer’s & Dementia 9 (2013) S105–S115

S113

Fig. 6. (A) Time course of VAb-related events after immunotherapy with 3D6 in PDAPP mice. (B) A model for vascular events after clearance of VAb by immunotherapy and a putative etiology of ARIA. In brief, (a) in the normal state, Ab is cleared via multiple pathways in the brain, including perivascular drainage. (b) Increased VAb deposition in AD leads to Ab accumulation in arterial walls. This deposition induces damage to the wall integrity and spontaneous transvascular leakage events may occur at a low rate. (c) When VAb is cleared after immunotherapy, the underlying impaired vasculature may be exposed, leading to fluid or blood leakage into the parenchyma, thereby creating the MRI abnormalities. Meanwhile, reduced perivascular clearance capacity may lead to the accumulation of Ab in capillaries and reduction in AQP4 water channel function. (d) With repeated treatment, persistent VAb clearance is expected to lead to recovery of blood vessel wall morphology and an elimination of the occurrence of new leakage events. At this same time, capillary Ab may be reduced, suggesting a recovery of perivascular capacity. Portions adapted from [37] with permission from John Wiley and Sons. Ó 2008 International Society of Neuropathology.

may be exposed, leading to a transient susceptibility period and possibly leading to fluid or blood leakage into the parenchyma, thereby creating the MRI abnormalities, ARIA-E and ARIA-H. Meanwhile, immunotherapyinduced increases in parenchymal Ab mobilization to a perivascular clearance pathway with reduced capacity may lead to the accumulation of Ab in capillaries. This newly deposited capillary Ab could then lead to a physical detachment of astrocytic endfeet from capillaries and a reduction in AQP4 channel function that slows the normally rapid clearance of extracellular fluid from the parenchyma. With chronic treatment, continued VAb clearance may lead to recovery of blood vessel wall integrity and an elimination of the occurrence of new leakage events (e.g., as evidenced by recovery of vascular morphology and reduction of microhemorrhages in the PDAPP mice). In parallel, capillary Ab may be reduced, suggesting recovery or increased compliance of perivascular capacity. Consistent with clinical data, this hypothetical model predicts that ARIA incidence should in-

crease under conditions that exacerbate Ab-induced vascular damage (e.g., as with APOE ε4 carriers) and with treatment approaches that increase the efficiency of VAb removal (e.g., as with high anti-Ab antibody dose). The model further predicts that such transvascular leakage events would be transient and ARIA should decrease with continued treatment, which has been noted in a few cases with continued bapineuzumab treatment [9]. Further consistent with this latter prediction is the observation that the neuropathological examination of patients immunized with AN1792 who died at various times after their first immunization demonstrated transiently increased CAA, with a significant involvement of capillary Ab and a higher density of cortical microhemorrhages [24]. Of note, patients that came to autopsy 4 to 5 years after their first immunization showed a relative absence of CAA, suggesting that, as observed in transgenic mice immunized with 3D6, increased CAA and microhemorrhages may have been transient events after AN1792 active immunization.

S114

W. Zago et al. / Alzheimer’s & Dementia 9 (2013) S105–S115

Collectively, the vascular amyloid clearance model of ARIA is consistent with preclinical and clinical data, including currently reported clinical risk factors and treatment response characteristics that have been described to date. Therefore, this model provides a framework for the generation of new hypotheses and the conduct of additional nonclinical and clinical studies aimed at furthering our understanding of the pathophysiology related to ARIA. Acknowledgments The authors gratefully acknowledge the expert technical assistance and contributions by Robin Barbour. The authors further acknowledge the expert technical assistance by Hong Yi at the Robert P. Apkarian Integrated Electron Microscopy Core at Emory University School of Medicine. All authors (except Karen Khan) were/are employees of Janssen Alzheimer Immunotherapy R&D, LLC. Wagner Zago, Sally Schroeter, Teresa Guido, Peter Seubert, Ted Yednock, Dale Schenk, Keith Gregg, Dora Games, Frederique Bard, and Gene Kinney were/are employees and/or shareholders of Elan Pharmaceuticals. Karen Khan was an employee of Janssen Alzheimer Immunotherapy R&D at the time of study conduct and was/is a shareholder of Elan Pharmaceuticals. This study was sponsored by Janssen Alzheimer Immunotherapy R&D, LLC, and Pfizer, Inc.

RESEARCH IN CONTEXT 1 Systematic review: The authors reviewed the literature using traditional (e.g., PubMed) sources and meeting abstracts and presentations. Although the pathophysiology of ARIA is not yet as widely studied as other aspects of AD biology, there have been several recent publications describing the clinical aspects of ARIA. These relevant citations are appropriately cited. 2 Interpretation: Our findings led to an integrated hypothesis describing the pathophysiology of ARIA. This hypothesis is consistent with nonclinical and clinical findings currently in the public domain. 3 Future directions: The manuscript proposes a framework for the generation of new hypotheses and the conduct of additional studies. Examples include further understanding 1) the role of perivascular clearance pathways on vascular changes after anti-Ab immunotherapy, 2) the role of alterations in water clearance mechanisms in the resolution of ARIA, 3) the potential reversibility of microhemorrhage events in the clinical setting, and 4) the relationship between the pathophysiology of ARIA-E and ARIA-H.

References [1] Hyman BT. New neuropathological criteria for Alzheimer disease. Arch Neurol 1998;55:1174–6. [2] Mangialasche F, Solomon A, Winblad B, Mecocci P, Kivipelto M. Alzheimer’s disease: clinical trials and drug development. Lancet Neurol 2010;9:702–16. [3] Schenk D, Games D, Seubert P. Potential treatment opportunities for Alzheimer’s disease through inhibition of secretases and Abeta immunization. J Mol Neurosci 2001;17:259–67. [4] Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H, et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med 2000;6:916–9. [5] Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, et al. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 1999;400:173–7. [6] Rinne JO, Brooks DJ, Rossor MN, Fox NC, Bullock R, Klunk WE, et al. 11C-PiB PET assessment of change in fibrillar amyloid-beta load in patients with Alzheimer’s disease treated with bapineuzumab: a phase 2, double-blind, placebo-controlled, ascending-dose study. Lancet Neurol 2010;9:363–72. [7] Sperling R. A randomized, double-blind, placebo-controlled clinical trial of intravenous bapineuzumab in patients with Alzheimer’s disease who are apolipoprotein E ε4 carriers. 16th Congress of the European Federation of Neurological Societies, Stockholm, Sweden, 2012. [8] Black RS, Sperling RA, Safirstein B, Motter RN, Pallay A, Nichols A, et al. A single ascending dose study of bapineuzumab in patients with Alzheimer disease. Alzheimer Dis Assoc Disord 2010;24:198–203. [9] Salloway S, Sperling R, Gilman S, Fox NC, Blennow K, Raskind M, et al. A phase 2 multiple ascending dose trial of bapineuzumab in mild to moderate Alzheimer disease. Neurology 2009; 73:2061–70. [10] Carlson C, Estergard W, Oh J, Suhy J, Jack CR Jr, Siemers E, et al. Prevalence of asymptomatic vasogenic edema in pretreatment Alzheimer’s disease study cohorts from phase 3 trials of semagacestat and solanezumab. Alzheimers Dement 2011;7:396–401. [11] Kloppenborg RP, Richard E, Sprengers ME, Troost D, Eikelenboom P, Nederkoorn PJ. Steroid responsive encephalopathy in cerebral amyloid angiopathy: a case report and review of evidence for immunosuppressive treatment. J Neuroinflammation 2010;7:18. [12] Sperling R, Bronen R, Greenberg SM, Sorensen G, Salloway S, Gass A, et al. Three cases of apparent vasogenic edema (VE) from a phase 2 clinical trial of the gamma secretase inhibitor BMS708163 in patients with mild-to-moderate AD. Alzheimers Dement 2011;7:S377. [13] Ostrowitzki S, Deptula D, Thurfjell L, Barkhof F, Bohrmann B, Brooks DJ, et al. Mechanism of amyloid removal in patients with Alzheimer disease treated with gantenerumab. Arch Neurol 2012; 69:198–207. [14] Sperling R, Salloway S, Brooks DJ, Tampieri D, Barakos J, Fox NC, et al. Amyloid-related imaging abnormalities in patients with Alzheimer’s disease treated with bapineuzumab: a retrospective analysis. Lancet Neurol 2012;11:241–9. [15] Sperling RA, Jack CR Jr, Black SE, Frosch MP, Greenberg SM, Hyman BT, et al. Amyloid-related imaging abnormalities in amyloid-modifying therapeutic trials: recommendations from the Alzheimer’s Association Research Roundtable Workgroup. Alzheimers Dement 2011;7:367–85. [16] Panza F, Frisardi V, Imbimbo BP, Seripa D, Paris F, Santamato A, et al. Anti-beta-amyloid immunotherapy for Alzheimer’s disease: focus on bapineuzumab. Curr Alzheimer Res 2011;8:808–17. [17] Zago W, Buttini M, Comery TA, Nishioka C, Gardai SJ, Seubert P, et al. Neutralization of soluble, synaptotoxic amyloid beta species by antibodies is epitope specific. J Neurosci 2012;32:2696–702.

W. Zago et al. / Alzheimer’s & Dementia 9 (2013) S105–S115 [18] Schroeter S, Khan K, Barbour R, Doan M, Chen M, Guido T, et al. Immunotherapy reduces vascular amyloid-beta in PDAPP mice. J Neurosci 2008;28:6787–93. [19] Seubert P, Barbour R, Khan K, Motter R, Tang P, Kholodenko D, et al. Antibody capture of soluble Abeta does not reduce cortical Abeta amyloidosis in the PDAPP mouse. Neurodegener Dis 2008;5:65–71. [20] Luna LG. Methods for pigments and minerals. In: Manual of histological staining methods of the Armed Forces Institute of Pathology. 3rd ed. New York: McGraw-Hill Book Co.; 1968. p. 174–88. [21] Wilcock DM, Rojiani A, Rosenthal A, Subbarao S, Freeman MJ, Gordon MN, et al. Passive immunotherapy against Abeta in aged APP-transgenic mice reverses cognitive deficits and depletes parenchymal amyloid deposits in spite of increased vascular amyloid and microhemorrhage. J Neuroinflammation 2004;1:24. [22] Burbach GJ, Vlachos A, Ghebremedhin E, Del Turco D, Coomaraswamy J, Staufenbiel M, et al. Vessel ultrastructure in APP23 transgenic mice after passive anti-Abeta immunotherapy and subsequent intracerebral hemorrhage. Neurobiol Aging 2007; 28:202–12. [23] Thal DR, Griffin WS, de Vos RA, Ghebremedhin E. Cerebral amyloid angiopathy and its relationship to Alzheimer’s disease. Acta Neuropathol 2008;115:599–609. [24] Boche D, Zotova E, Weller RO, Love S, Neal JW, Pickering RM, et al. Consequence of Abeta immunization on the vasculature of human Alzheimer’s disease brain. Brain 2008;131:3299–310. [25] Papadopoulos MC, Manley GT, Krishna S, Verkman AS. Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema. FASEB J 2004;18:1291–3. [26] Wilcock DM, Vitek MP, Colton CA. Vascular amyloid alters astrocytic water and potassium channels in mouse models and humans with Alzheimer’s disease. Neuroscience 2009;159:1055–69. [27] Yang J, Lunde LK, Nuntagij P, Oguchi T, Camassa LM, Nilsson LN, et al. Loss of astrocyte polarization in the tg-ArcSwe mouse model of Alzheimer’s disease. J Alzheimers Dis 2011;27:711–22. [28] Attems J, Yamaguchi H, Saido TC, Thal DR. Capillary CAA and perivascular Abeta-deposition: two distinct features of Alzheimer’s disease pathology. J Neurol Sci 2010;299:155–62.

S115

[29] Wilcock DM, Colton CA. Immunotherapy, vascular pathology, and microhemorrhages in transgenic mice. CNS Neurol Disord Drug Targets 2009;8:50–64. [30] Racke MM, Boone LI, Hepburn DL, Parsadainian M, Bryan MT, Ness DK, et al. Exacerbation of cerebral amyloid angiopathyassociated microhemorrhage in amyloid precursor protein transgenic mice by immunotherapy is dependent on antibody recognition of deposited forms of amyloid beta. J Neurosci 2005;25:629–36. [31] Rigol M, Solanes N, Roque M, Farre J, Batlle M, Roura S, et al. Hemosiderin deposits confounds tracking of iron-oxide-labeled stem cells: an experimental study. Transplant Proc 2008;40:3619–22. [32] Cordonnier C, van der Flier WM. Brain microbleeds and Alzheimer’s disease: innocent observation or key player? Brain 2011;134:335–44. [33] Biffi A, Greenberg SM. Cerebral amyloid angiopathy: a systematic review. J Clin Neurol 2011;7:1–9. [34] Nagy Z, Esiri MM, Jobst KA, Johnston C, Litchfield S, Sim E, et al. Influence of the apolipoprotein E genotype on amyloid deposition and neurofibrillary tangle formation in Alzheimer’s disease. Neuroscience 1995;69:757–61. [35] Schmechel DE, Saunders AM, Strittmatter WJ, Crain BJ, Hulette CM, Joo SH, et al. Increased amyloid beta-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease. Proc Natl Acad Sci U S A 1993;90:9649–53. [36] Preston SD, Steart PV, Wilkinson A, Nicoll JA, Weller RO. Capillary and arterial cerebral amyloid angiopathy in Alzheimer’s disease: defining the perivascular route for the elimination of amyloid beta from the human brain. Neuropathol Appl Neurobiol 2003;29:106–17. [37] Weller RO, Subash M, Preston SD, Mazanti I, Carare RO. Perivascular drainage of amyloid-beta peptides from the brain and its failure in cerebral amyloid angiopathy and Alzheimer’s disease. Brain Pathol 2008;18:253–66. [38] Weller RO, Preston SD, Subash M, Carare RO. Cerebral amyloid angiopathy in the aetiology and immunotherapy of Alzheimer disease. Alzheimers Res Ther 2009;1:6. [39] Tarkowski E, Liljeroth AM, Minthon L, Tarkowski A, Wallin A, Blennow K. Cerebral pattern of pro- and anti-inflammatory cytokines in dementias. Brain Res Bull 2003;61:255–60.