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Atherosclerosis 210 (2010) 78–87

Contents lists available at ScienceDirect

Atherosclerosis

journa l homepage: www.e lsev ier .com/ locate /a therosc leros is

ncreased atherosclerosis and vascular inflammation in APP transgenicice with apolipoprotein E deficiency

. Tibollaa,b,1, G.D. Norataa,b,∗,1, C. Medaa,c, L. Arnaboldia, P. Uboldia, F. Piazzad,. Ferraresed, A. Corsinia, A. Maggia,c, E. Vegetoa,c, A.L. Catapanoa,b,e,∗∗

Department of Pharmacological Sciences, Università degli Studi di Milano, Milan, ItalySISA Center for the Study of Atherosclerosis, Bassini Hospital, Cinisello Balsamo, ItalyCenter of Excellence on Neurodegenerative Diseases, Department of Pharmacological Sciences, Università degli Studi di Milano, Milan, ItalyDepartment of Neuroscience and Biomedical Technology, University of Milano-Bicocca, Monza, ItalyI.R.C.C.S. MultiMedica Hospital, Milan, Italy

r t i c l e i n f o

rticle history:eceived 17 April 2009eceived in revised form 29 October 2009ccepted 30 October 2009vailable online 10 November 2009

eywords:polipoprotein E

a b s t r a c t

Objective: Atherosclerosis is associated with Alzheimer’s disease (AD) in humans, but the nature of thislink is still elusive. Aim of this study was to investigate aortic atherosclerosis development in a mousemodel with central nervous system (CNS) restricted �-amyloid precursor protein (APP) overexpression.Methods and results: APP23 mice, overexpressing the Swedish mutated human APP selectively in the brain,were crossed with mice lacking apolipoprotein E (ApoE KO). Nine weeks old mice were fed a westerntype diet for eight weeks, then atherosclerotic lesions, aortic wall and cortical tissues gene expressionand �-amyloid (A�) deposition were evaluated.

myloid � proteinascular inflammation

Compared with ApoE KO, APP23/ApoE KO mice developed larger aortic atherosclerotic lesions andshowed significantly increased expression of MCP-1, IL-6, ICAM-1 and MTPase 6, a marker of oxida-tive stress in the vascular wall. Of note brain limited APP synthesis was associated with an increasedmicroglia and brain endothelial cells activation, in spite of the absence of �-amyloid deposits in the brainor alteration in the levels of oxidized metabolites of cholesterol such as 4-cholesten-3-one.Conclusion: Our study suggests that the vascular pro-inflammatory effects of CNS-localised APP overex-

nesis

pression lead to atheroge

. Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerativeisorder characterised by extracellular A� peptide deposition inhe senile plaques and intracellular formation of neurofibrillaryangles, consisting of aggregated, hyperphosphorilated tau protein,ccompanied by neuron loss, finally leading to dementia [1].

An increasing body of evidence suggests the presence of a linketween AD and cardiovascular diseases, especially atherosclerosis2]. The presence of AD neuropathology hallmarks in many casesf non-demented coronary artery disease (CAD) at post mortem,

∗ Corresponding author at: Department of Pharmacological Sciences, Universitàegli Studi di Milano, Via Balzaretti 9, 20133 Milan, Italy. Tel.: +39 02 50318293;ax: +39 02 50318386.∗∗ Corresponding author at: Department of Pharmacological Sciences, Universitàegli Studi di Milano, Via Balzaretti 9, 20133 Milan, Italy. Tel.: +39 02 50318302;ax: +39 02 50318386.

E-mail addresses: Danilo.Norata@unimi.it (G.D. Norata),lberico.Catapano@unimi.it (A.L. Catapano).1 Equally contributed.

021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.atherosclerosis.2009.10.040

before parenchymal A� deposition and neuronal dysfunction.© 2009 Elsevier Ireland Ltd. All rights reserved.

led researchers to consider CAD as a risk factor for development ofAD [3]. Hypercholesterolemia and inflammation are the dominantmechanisms implicated in the development of atherosclerosis andhave emerged as key factors implicated in the Alzheimer’s diseasepathogenesis as well [4].

The possible implication of altered cholesterol homeostasis inAD is documented by several studies showing that alteration inbrain cholesterol levels affects A� formation from amyloid pre-cursor protein (APP) [5,6]. More recently, A� has been shown tobind Cu2+ with very high affinity, forming a redox-active complexwhich selectively oxidizes cholesterol at the C-3 hydroxyl group,catalytically producing 4-cholesten-3-one [7], a metabolite highlyexpressed in brain from AD patients and from APP transgenic mice[7].

However, while the influence of impaired cholesterol homeosta-sis on AD has been recognised, the effects of �-amyloidosis and

neurodegeneration on peripheral atherosclerosis are unclear. APPand A� peptides are present in advanced human carotid plaques, inproximity to activated macrophages and platelets [8] and the devel-opment of non-dietary induced early atherosclerotic lesions hasbeen recently observed in A� transgenic mice [9]. Taken together,

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hese findings support the hypothesis that atherosclerosis and ADre independent but convergent diseases [10].

Aim of this work was to investigate whether CNS restrictedPP overexpression could affect atherosclerosis and vascular wall

nflammation, by generating a double transgenic mice with humanutated APP overexpression restricted to neurons [11], combinedith apolipoprotein E deficiency.

. Materials and methods

.1. Animals and diets

ApoE KO mice were purchased from Jackson laboratoriesBar Harbor, ME, USA), APP23 mice overexpressing Swedish

utated (K670N/M671L) human APP (hAPP751) under the reg-latory control of the neuron-specific murine Thy-1 promoter11] were kindly provided by Dr. Matthias Staufenbiel, Novar-is Pharma AG, Basel, Switzerland. The mice were on C57Bl/6Jackground and were crossed to obtain the double trans-enic line designated as APP23/ApoE KO. The transgenic statusf each animal was confirmed by polymerase chain reactionPCR) from tail DNA using specific primers for human APP5′-GAATTCCGACATGACTCAGG-3′ and 5′-GTTCTGCATCTTGGACA-′ forward and reverse primer respectively). The genotyping forpoE has been performed following the instructions described

n the Jackson Laboratory web site (http://jaxmice.jax.org/pub-gi/protocols/protocols.sh?objtype=protocol&protocol id=221).

Nine weeks old APP23/ApoE KO mice, ApoE KO, APP23 and57Bl/6J (10 animals for each group, 5 male and 5 female) were

ed an atherogenic western type diet (21% fat, 0.15% cholesterolnd 19.5% casein, Piccioni, Italy) for eight weeks. The mice wereacrificed with intraperitoneal injection of Avertin 2.5% (Aldrichhemical Co., USA), according to the protocol of the internal ethicalommittee (Department of Pharmacological Sciences, University ofilan). The hearts and the aortas were quickly removed and fixed

n 3% neutral buffered formalin solution and paraffin embeddedapproximately from the valve leaflets up to the first part of thescending aorta) or placed in RNAlaterIce (Ambion, Germany) at20 ◦C for the RNA isolation [12]. The right hemispheres of therains were processed for the isolation of cortex and hippocam-us. The left hemispheres were snap-frozen in liquid nitrogen andtored at −80 ◦C until analysed [13].

For RNA isolation the samples were homogenised in a dis-embrator (B. Braun, Melsungen AG, Germany) then processed as

escribed [14]. All experimental procedures were in accordanceith the institutional guidelines for animal research.

.2. Determination of plasma lipids concentrations andipoprotein cholesterol profile

Blood samples were collected in 5% EDTA tubes from anes-hetized animals by retro-orbital bleeding and plasma waseparated by low speed centrifugation at 4 ◦C. The measurementsf plasma lipids was performed by standard enzymatic techniquesABX for Cobas Mira Plus, Montpellier, France) as described [12].he 1.006–1.21 (g/ml) density fraction was isolated followingBr gradient density ultracentrifugation, and plasma lipoproteinholesterol profiles were analysed by fast protein liquid chro-atography (FPLC) [15].

.3. Characterization of atherosclerotic lesions

Cross-sequential 5 �m sections were collected on slides fromhe aortic root up to 250 �m until the valve leaflets disappear. Sec-ions of the aortic sinus [12] (a total of 10 sections, one every 25 �m)ere stained with hematoxilin and counterstained with eosin; then

sis 210 (2010) 78–87 79

the images were captured with Axiovert 200 microscope (ZEISS,Milan, Italy) and the atherosclerotic lesion area was quantified bycomputer image analysis, using the Axiovision LE rel 4.4 software byan observer blinded to the nature of the samples. Data are expressedas �m2 ± standard deviation.

The quantification of the collagen content was performed fol-lowing picro-sirius red and Van Gieson staining, images of theaortas were captured with Axiovert 200 microscope (ZEISS) underregular and polarized light, then fibrillar collagen, detected by yel-low birefringence, was quantified using Image J analysis software,capable of colours segmentation and automation by programmablemacros, by two blinded observers. Data are expressed as the per-centage of the total atherosclerotic lesion area covered by fibrillarcollagen ± standard error.

2.4. Amyloidˇ quantification by ELISA

Fresh frozen hyppocampi were homogenised in a dismembra-tor (B. Braun, Melsungen AG, Germany) in HEPES/Na 25 mM, EDTA2 mM, EGTA 1 mM buffer pH 7.4 containing protease inhibitors(Complete protease inhibitor cocktail, 1 tablet in 10 ml solution,Roche, Germany) and centrifuged for 10 min at 4 ◦C at 1000 × g.Then the pellet was removed and the supernatant further cen-trifuged at 100,000 × g for 1 h at 4 ◦C and the final supernatantswere used to measure water soluble A�1–42 peptide. The proteincontent was quantified by the Lowry assay. For ELISA detection ofthe A�1–42 peptide the human A�1–42 kit (IBL, Hamburg, Germany)was used according to the manufacturer protocol. The assay has ahigh-cross reactivity for mouse/rat A�1–42 (70.9%), therefore val-ues observed in control animals (C57Bl/6J and Apo E KO) possiblyreflect the levels of endogenous A�1–42 peptide. The final resultswere adjusted to ng per mg of protein.

2.5. Real-time quantitative polymerase chain reaction

Total RNA was extracted and processed for reverse transcription,then RT-PCR or Q-PCR experiments were performed as described[16,17]. Briefly 3 �l of cDNA were amplified using 1 �M primersspecific for human APP, 5′-AGGAAGCAGCCAACGAGAG-3′ and 5′-GCAGAGCGGTGATGTAGTTC-3′, 10 �M dNTPs, TaQ Pol (Promega,Milan, Italy) with the following thermal protocol: 7′ at 95 ◦C and30′ at 95 ◦C, 40′ at 60 ◦C for 25 cycles. For each sample a con-trol reaction with primers to the RLP13a (housekeeping gene), wasrun as quality control. For quantitative real-time PCR experiments3 �l of cDNA were amplified using 1× Sybr Greener mastermix(Invitrogen, Italy). The specificity of the Sybr green fluorescencewas tested by amplicons melting curves as described [13]. Theprimers used were described previously [12] with the exceptionof primers for IL1�, MIP2 and TNF alpha and were all from Assayon Demand (Applera, Italy). Each sample was analysed in duplicateusing the IQTM thermal cycler (BioRad, Italy), the PCR amplificationwas related to a standard curve ranging from 10−11 to 10−14 mol/land data were normalized for the housekeeping gene ribosomalprotein 13a (RLP13a).

2.6. Immunohistochemical analysis of cerebral and aortic tissues

For immunohistochemical analysis of cerebral tissues, freshfrozen hemibrain sagittal sections, 30 �m thick, were collectedusing a cryostat (Microm, Waaldorf, Germany), whereas 5 �mthick sections were collected from paraffin-embedded aortic tis-

sues as described above. Three sections per animal were stainedon gelatin-coated microscope slides (Menzel-Glaser). The sectionswere washed for 2 min in PBS (for Mac-1 staining, the sectionswere pre-washed with NHCl4 0.05 M for 30 min, for human APPand A� staining were incubated 15 min at room temperature in

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ormic acid 95% prepared in Ethanol 100%, for paraffin embed-ed tissues antigen retrieval was performed by treatment withroteinase K solution (20 �g/ml) for 10 min at 37 ◦C), then formin in distilled water and three times in PBS then pre-treatedmin in 1% H2O2 at room temperature to inhibit endogenouseroxidases and blocked in blocking solution (10% normal goaterum, 0.1% Triton X-100 in PBS) or alternatively with 5% non-fatilk in PBS/Tween 0.1% for 1 h at room temperature. The tissue

ections were incubated with the following primary antibodies:myloid�/Amyloid A4 epitope specific (aa 8–17 of human amy-

oid � protein) polyclonal rabbit antibody, Neo Markers, Lab Visionorporation, Fremont, CA, USA, 0.4 �g/ml in 10% normal goat serumnd PBS; Mac-1, Cd11b specific rat antibody, AbD Serotec, Oxford,K; Rat anti mouse F4/80 antigen, AbD Serotec, Oxford, UK; Rabbitolyclonal to CD3 and rabbit polyclonal to smooth muscle actin,bcam, Cambridge UK; Rat anti mouse CD 31, BD Biosciences,rance; Goat anti-ICAM-1, R&D systems, Minneapolis, MN, USA;oat polyclonal anti 8-hydroxy-guanosin, Abcam or non-immune

gG as a control, overnight at 4 ◦C, followed by incubation with aecondary biotinylated antibody (anti-rabbit IgG, or anti-rat IgGr anti-goat IgG, Vector Laboratories, Burlingame, CA, USA, diluted:200 in 1% normal goat serum and PBS) 1 h at room temperature.mmunoreactivity was visualised using avidin-biotin-peroxidase

ethod (Vectastain ABC kit from Vector Laboratories, Burlingame,A, USA) with 3,3′-diaminobenzidine substrate (Sigma, Italy) as thehromogen.

Dehydrated sections were observed using Zeiss Axioskop micro-cope (Zeiss) and analysed with color-video image analysis systeminked to the microscope. For the quantification of the immunore-ctive area the Image J analysis software, capable of colourseconvolution and segmentation by programmable macros, wassed. Data are expressed as the percentage of the total atheroscle-otic lesion area ± standard error.

.7. Lipid extraction

Mouse hemibrains were thawed from −80 ◦C, put in glass vialsn a proper volume of chloroform/methanol 2:1 plus BHT 0.01%s antioxidant, and homogenized by a polytron and, lipids werextracted according to Folch. A known amount of stigmasterol wasdded in each sample as an internal standard. In detail, after addi-ion of KCl 0.05%, the vials were shaken overnight centrifuged at.500 rpm for 15 min, and the bottom phase retrieved. The organichases were merged in a glass vial, dried in a stream of nitrogennd resuspended in chloroform/methanol 2:1 plus BHT. After thextraction, the aqueous supernatant phase was dried, the result-ng pellet dissolved with NaOH 0.1N and the total protein amountuantified utilizing the bicinchoninic acid (BCA) assay. All theesults were normalized by the protein content and expressed asg/mg protein. Vials, standards and reagents were purchased fromigma–Aldrich (Milano, Italy).

.8. Free cholesterol and 4-cholesten-3-one analysis

An aliquot of the lipid extract was loaded onto a channeled Sil-ca TLC plate (BioMap, Italy) prerun in hexane/diethyl ether/aceticcid 87:20:1 (v/v/v), heat-activated and developed up to 2.5 cmrom the top. Corresponding blank lanes were also loaded withhloroform/methanol. After run, the TLC plates were sprayed withichlorofluorescein (0.15% in ethanol) and, after drying in a streamf nitrogen, the silica spots corresponding to those of the known

tandards of free cholesterol, stigmasterol and 4-cholesten-3-oneere scraped off. It is noteworthy to mention that in our experi-ental conditions we were not able to detect any macroscopically

isible spot corresponding to 4-cholesten-3-one in any sample.terols were subsequently extracted twice with hexane/water 1:6

sis 210 (2010) 78–87

after centrifugation at 2500 rpm for 15 min. An aliquot of each sam-ple, after being concentrated in a stream of nitrogen to preventoxidation, was loaded without derivatization on a DANI 1000 gasliquid chromatographer (GLC) (DANI instruments, Milano, Italy)equipped with a flame ionization detector, an autosampler andfitted with a 30 m, 0.32 mm, 0.25 �m MEGA-1 (Mega Columns, Leg-nano, Italy) fused silica column. An example of the run of knownunderivatized standards and a representative run of a brain sam-ple are depicted in Supplementary Figure I. GLC parameters wereas follows: flow of hydrogen at a constant pressure of 1 bar; oventemperature from 185 ◦C to 300 ◦C (at a gradient of 10 ◦C/min) andmaintained at this temperature for 5 min. The chromatograms ofeach sample have been recorded and the area of each peak quanti-fied by the dedicated Clarity Software (Clarity, Prague, Czechia). Themass of each substance was calculated by comparison with that ofthe internal standard (stigmasterol) and results were normalizedby the protein content of each sample.

2.9. Statistics

Data were analysed using SPSS 16.0 for windows (SPSS, Chicago,IL). Statistical analysis was performed with Student T-test, settingthe significance level at p < 0.05.

3. Results

3.1. Human APP expression in APP23/ApoE KO mice

To evaluate the tissue specificity in the expression of the hAPP751transgene in the APP23/ApoE KO mouse model, we investigatedhuman APP expression in cerebral and peripheral tissues. To thispurpose cDNA samples from liver, lung, adipose tissue, aorta, heartand cerebral cortex were amplified by PCR using specific primers forhuman APP mRNA. An amplification product of the expected lengthwas obtained from cortex of APP23/ApoE KO animals, whereashuman APP gene expression was not detected either in other tissuesor in cortical tissues from ApoE KO mice (Fig. 1A and B). Accord-ingly, intracellular granular deposits of A� were observed in corticalneurons of APP23/ApoE KO animals (Fig. 1C and E) and were notassociated with any alteration in neuronal morphology or number,as originally described for the parental APP23 line [18]. As expected,the A� immunostaining in ApoE KO resulted in a diffuse signal dueto endogenous murine APP cross-reactivity (Fig. 1D and F). A sim-ilar pattern was observed in C57Bl/6J wild-type animals, while A�deposits were detected as cored plaques in 12-months-old APP23mice (Fig. 1G and H). These observations indicate that the lack ofApoE and the concomitant hypercholesterolemia do not affect A�deposition and neuron viability in 4-months-old APP23/ApoE KOanimals.

3.2. Aˇ-peptides overexpression, neuroinflammation and brainendothelial activation

Next we investigated whether CNS-restricted APP overexpres-sion was associated with neuroinflammation. First, we assayedthe activation state of microglia cells, the resident macrophagecells of the brain, which reflects an ongoing inflammatory reactionand that can be observed by both biochemical and morphologicalparameters. We analysed the expression of Mac-1 (CD11b), a mem-brane protein up regulated in macrophages by a variety of stimuliand involved in the brain inflammatory response. Microglia cells

in the hippocampus and frontal cortex of APP23/ApoE KO miceshowed an increased expression of Mac-1 compared to ApoE KOmice (Fig. 2A–C). Nevertheless, the morphology of microglia cellswas only mildly different from that of wt (not shown) or ApoEKO mice, with a slight increased in cell body size and branchings.

G. Tibolla et al. / Atherosclerosis 210 (2010) 78–87 81

Fig. 1. Expression of hAPP mRNA (panel A and B) and of A� deposition in APP23/ApoE KO and ApoE KO mice. Panel A and B: mRNA expression for hAPP and RLP 13a(housekeeping gene) was measured by means of RT-PCR. The results derive from 7 pooled APP23/Apo E KO and 5 pooled Apo E KO animals. For each group, the expression inthe liver, lung, adipose tissue, aorta, heart and cerebral cortex was studied. The experiment was repeated twice and a representative panel is shown. Panel C–H: serial frozenh d 12-m� ngulat oup is

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emibrain sagittal sections from APP23/ApoE KO (C and E), ApoE KO (D and F) an/Amyloid A4 polyclonal epitope specific antibody. A representative image of the ci

he area magnified in E and F) and a magnification of cortical neurons from each gr

ully activated macrophages instead display shorter and thickerell processes and a round-shaped cell body. We thus evaluated thexpression of inflammatory genes in the cortex and hippocampus.he mRNA levels of MCP-1, IL-6, inducible nitric oxide synthaseiNOS), IL-1�, were not different comparing APP23/ApoE KO andpoE KO transgenic mice, while MIP-2 and TNF alpha mRNA expres-ion was slightly elevated (Fig. 2D). Next we studied whetherdhesion molecules expression was affected in the brain. IncreasedCAM-1 mRNA levels were detected in APP23/ApoE KO comparedo ApoE KO mice, while no differences were observed for VCAM-

mRNA expression (Fig. 2D). It is interesting to underline thatCAM-1 is a ligand of the integrin complex containing Mac-1 [19],

hich might provide a link between the increase in these twonflammatory markers. These findings were confirmed by immuno-istochemistry; blood vessels were first detected by CD31 staining

onths-old APP23 (G) and C57Bl/6J (H) mice were immunostained with Amyloidted cortex proximal to corpus callosum is presented in C and D (the arrows indicateshown in E–H. Scale bar for C and E: 200 �m and for E–H: 10 �m.

and then ICAM-1 expression was investigated. APP23/ApoE KObrains showed increased expression of ICAM-1 compared to ApoEKO brains (Fig. 2E–L). These results suggest that APP overexpressionin ApoE KO mice is associated with initial signs of brain macrophageand endothelial activation.

The expression of amylodogenic peptides per se could explainthe observed endothelial activation and indeed several authorsreported that A� peptides induce the expression of adhesionmolecules as well as of other inflammatory mediators in brainendothelial cells, thus leading to endothelial dysfunction [20,21].

We also investigated additional mechanisms that might be poten-tially involved. First we tested whether the overexpression of APPwas associated with an increased oxidative stress; the expressionof MTPase6, a key enzyme involved in the mitochondrial oxida-tive chain [22], was slightly but not significantly increased in the

82 G. Tibolla et al. / Atherosclerosis 210 (2010) 78–87

Fig. 2. Microglial activation, neuroinflammatory mediators and adhesion molecules expression in brains. Panel A–C: serial frozen hemibrain sagittal sections from APP23/ApoEKO (A and B) and from ApoE KO mice (C) were immunostained with non-immune IgG (A) or with an antibody specific for Mac-1 (B and C). A representative image from eachgroup is shown. Scale bar: 200 �m. Panel D: relative mRNA expression (ApoE KO = 1) of MCP-1, IL-6, iNOS, IL1�, MIP-2, TNF alpha, ICAM-1 and VCAM-1 is shown (n = 8 foreach group). Results, corrected for the RLP13a expression, are shown as fold of induction vs. ApoE KO (data are expressed as mean ± S.E.). Panel E–L: serial frozen hemibrains stainei froms

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agittal sections from APP23/ApoE KO (E–G) and from ApoE KO (H–L) were immunon panel E and G represents the area magnified in F,G and I,L. A representative imageignals colocalise). Scale bar: 100 �m.

rain of APP23/ApoE KO compared to ApoE KO (Fig. 3A); the lev-ls were instead significantly increased in the aorta, suggestinghat oxidative stress involving the mitochondrial chain could behe consequence of the on-going atherogenic process. Similarly,he expression of 8-hydroxy-guanosine, a marker of DNA oxida-

ion was unchanged between APP23/ApoE KO and ApoE KO miceSupplementary Figure II). Next, since A� is known to generateredox-active complex which oxidizes cholesterol selectively at

he C-3 hydroxyl group, catalytically producing 4-cholesten-3-one7], we tested the hypothesis that the increased atherosclerosis

d with an antibody specific for CD31 (E, F and H, I) or ICAM-1 (G and L). The squareeach group is shown (the arrows indicate the area where the CD31 and the ICAM-1

could be the consequence this oxidative process. As detected bygas-chromatography (Supplementary Figure I), 4-cholesten-3-oneconcentrations in the brain were very low and were not differentbetween APP23/ApoE KO and ApoE KO (Fig. 3B). Also the con-centrations of free cholesterol, which accounts for 99.5% of brain

cholesterol [23], despite elevated, were not significantly changedbetween APP23/ApoE KO and ApoE KO (Fig. 3B). These observa-tions seem to exclude two possibilities: first, that the mouse modelhere studied presents additional (increased) free cholesterol thatcould be susceptible to modification and second, that at least prior

G. Tibolla et al. / Atherosclerosis 210 (2010) 78–87 83

Fig. 3. Oxidative stress in the brain and amyloid � deposition in aorta of APP23/ApoE KO and ApoE KO mice. Panel A: relative mRNA expression (ApoE KO = 1) of MTPase6 isshown for brain and aortae (n = 8 for each group). Results, corrected for the RLP13a expression, are shown as fold of induction vs. ApoE KO (data are expressed as mean ± S.E.).P /ApoEw 23/Aps ith heA C–E) a

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anel B: 4-cholesten-3one and free cholesterol concentrations in brain from APP23ere studied). Panel C–F: serial sections of ascending aorta from ApoE KO and APP

pecific antibody (panel D–F) or non-immune IgG (panel C), and counterstained wPP23 1 year old mice (panel F) used as a positive control. Scale bar: 20 �m (figure

o deposition in brain, A� peptides may increase the generationf 4-cholesten-3-one, an oxidized metabolite of cholesterol withytotoxic properties. However we cannot exclude the possibilityhat other oxidized lipid metabolites could play a role in promotingndothelial dysfunction.

Since ApoE genetic ablation results in reduced amyloid deposi-ion, possibly through its increased clearance from the brain [24],e investigated A� brain, and plasma levels and A� deposition in

he aorta of ApoE KO and APP23/ApoE KO mice. As expected A� pep-ides levels were significantly higher in the brain of APP23/ApoE KOompared to ApoE KO (Table 1); similar results were obtained when� peptides levels were studied in brain from APP23 and C5Bl/6J

Table 1). Using the ELISA assay approach we did not observe theresence either of A� peptides in the plasma of mice (data nothown) or A� immunoreactivity within the atherosclerotic plaquef the mice (Fig. 3C–F). These results suggest that brain-derived A�ould directly stimulate the vascular inflammation and atheroscle-osis observed in APP23/ApoE KO independently of its depositionithin the atherosclerotic plaque.

.3. Body weight, plasma lipids and lipoprotein cholesterol profile

We then evaluated the effects of human APP overexpressionn body weight, plasma lipids and lipoprotein cholesterol profile.

KO and ApoE KO mice. Data are presented as mean ± S.E. (5 animals for each groupoE KO mice were immunostained with Amyloid �/Amyloid A4 polyclonal epitopematoxilin and eosin. A� deposits are detected as cored plaques only in brain fromnd 160 �m (panel F).

Nine weeks-old mice were fed for eight weeks a western type dietand their body weight was recorded weekly. No significant differ-ences were observed in the weight gain between the two groupsfor both male and female animals (data not shown). Total choles-terol and triglycerides were slightly but not significantly increasedin APP23/ApoE KO mice compared to ApoE KO mice (Table 1).FPLC lipoprotein cholesterol profiles showed no differences in thecholesterol distribution between APP23/ApoE KO and ApoE KOmice. VLDL and LDL were increased and HDL decreased comparedto C57Bl/6J mice fed a normal chow diet in both groups, as expectedafter feeding an atherogenic diet (data not shown).

3.4. Characterization of atherosclerosis in APP23/ApoE KO mice

We then investigated the extension and the characteristics ofthe aortic atherosclerotic lesions. Morphometric analysis showedthat APP23/ApoE KO mice developed significantly larger aorticatherosclerotic lesions compared to ApoE KO mice, with a 41%increase of the lesion size observed in the aortic sinus (p < 0.05)

(Fig. 4A and B; Table 1). Female mice have larger atheroscle-rotic lesions compared to males, independent of the presenceof APP23/ApoE KO mice (data not shown). When the thoracicand the abdominal tracts of the aorta were investigated no rele-vant atherosclerotic lesions were observed (Supplementary Figure

84 G. Tibolla et al. / Atherosclerosis 210 (2010) 78–87

Table 1Plasma lipids, aortic atherosclerotic lesions and brain A�1–42 quantification.

C57Bl/6J (n = 10) APP23 (n = 10) ApoE KO (n = 10) APP23/ApoE KO (n = 10)

Plasma total Cholesterol (mg/dl) 144 ± 30 119 ± 26 943 ± 270 1,098 ± 213Triglycerides (mg/dl) 52 ± 12 46 ± 11 104 ± 43 126 ± 80Aortic sinus lesion (�m2) n.d. n.d. 156,464 ± 13,947 220,026 ± 23,888*

Brain A�1–42 (ng/mg protein) 73 ± 20 108 ± 27§ 69 ± 14 115 ± 27*

Nine weeks old mice were fed a western type diet for 8 weeks. Data are expressed as mea* p < 0.05 vs. ApoE KO.§ p < 0.05 vs. C57Bl/6J.

Fig. 4. Atherosclerosis in APP23/ApoE KO and ApoE KO mice. Representative sec-tions of aortic sinus (panel A) stained with hematoxilin and eosin, from ApoE KOand APP23/ApoE KO female mice are shown, scale bar: 100 �m. The extent of theatherosclerotic lesion in ApoE KO and APP23/ApoE KO mice for aortic sinus is shownin panel B (♦= male and © = female). Data (n = 10 for each group) are expressed asmean ± S.E. (*p < 0.05 APP23/ApoE KO vs. ApoE KO).

Fig. 5. Fibrillar collagen within the atherosclerotic lesion. Representative sections of aorpresented as the percentage of atherosclerotic lesion covered by sirius red positive fibrillthe area positive for fibrillar collagen in APP23/ApoE KO and ApoE KO mice (n = 10 for eachas mean ± S.E.

n ± S.E. n.d. mean: not detectable.

IV). Since no atherosclerotic lesions were detected in APP23mice and C5Bl/6J mice upon eight weeks of western type diet,(Supplementary Figure III and Table 1) we focused our next analysisonly on double transgenic mice.

We also investigated the collagen content within the lesionsand no differences were observed for fibrillar collagen depositionbetween APP23/ApoE KO and ApoE KO mice following picro-siriusred or Van Gieson stainings (Fig. 5 and Supplementary Figure V),thus suggesting a similar extracellular matrix deposition. When weevaluated the presence of macrophages, smooth muscle cells andT lymphocytes in the atherosclerotic lesions, no differences wereobserved for smooth muscle cells and T lymphocytes, while thepresence of macrophages was significantly higher in APP23/ApoEKO compared to ApoE KO mice (Fig. 6). Since localised chronicinflammation has been shown to influence the development andthe progression of atherosclerosis [25], we studied the expres-sion of pro-inflammatory and anti-inflammatory mediators in thearterial wall of APP23/ApoE KO mice, to investigate whether theincreased presence of macrophages in APP23/ApoE KO could beassociated with an increased inflammatory response.

Quantitative real-time PCR showed a significantly increasedexpression of macrophage chemoattractant protein 1 (MCP-1) andinterleukin 6 (IL-6) in APP23/ApoE KO compared to ApoE KO mice,while no differences were observed in the expression of other genessuch as MIP1-�, IL-10 and of the chemokines receptors CCR1 andCCR2 (Fig. 7A). Finally we investigated the expression of adhe-sion molecules in the vessel wall and we observed an increasedexpression of intracellular adhesion molecule 1 (ICAM-1) mRNA(Fig. 7A); while a slight but not significant difference was observedfor VCAM-1 mRNA expression (Fig. 7A). This observation was con-firmed by immunocytochemistry, where increased ICAM-1 levels

were detected not only in the aorta but also in other vesselssuch as coronary arteries (Fig. 7B–I). These results suggest that anincreased inflammatory response is present in the vascular wall ofAPP23/ApoE KO mice compared to ApoE KO mice; and is associatedwith increased atherosclerosis.

tic sinus stained with sirius red from APP23/ApoE KO and ApoE KO. The data arear collagen and are expressed as mean ± S.E., scale bar: 100 �m. The percentage ofgroup) for aortic sinus is shown on the right part of each panel. Data are expressed

G. Tibolla et al. / Atherosclerosis 210 (2010) 78–87 85

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ig. 6. Relative expression of T lymphocytes, smooth muscle cells and macrophages inalysis (Panel B) of T-lymphocytes, smooth muscle cells and macrophages. Data replaque. *p < 0.05 vs. ApoE KO. A 5× magnification is presented in the upper panels,

. Discussion

The present study shows that CNS specific amyloidosis pro-otes endothelial activation, accelerates atherosclerotic lesion

evelopment and vascular inflammation in the arterial wall of micerone to atherosclerosis.

Atherosclerosis is associated with amyloidosis and AD inumans, and brain microvasculopathies have been shown to corre-

ate with aortic atherosclerosis in patients with hereditary cerebralaemorrhage with amyloidosis Dutch type [26].

An open question pertains to the specific contribution of A�eptides produced in the brain and accumulated into the senilelaques after oligomerization and aggregation. A positive correla-

therosclerotic plaque. Representative photomicrographs (Panel A) and quantitativethe percentage (mean ± S.E.) of positive immunoreactive area of the atheroscleroticmes identify areas that are magnified (20×) in the lower panels.

tion between cerebral A� load and aortic fatty streak formation hasbeen reported by Li et al. [9] in a mouse model where the humanAPP transgene is under the control of the hamster prion protein(PrP) promoter and widely expressed in peripheral tissues. In ourstudy, we aimed at extending these observations by investigatingthe effect of cerebral localised amyloidosis on aortic atheroscle-rosis and on the inflammatory response. To address this issue,we generated a transgenic mouse line with an overproduction of

A� selectively in neurons on an atherosclerosis prone background(ApoE KO).

In APP23/ApoE KO mice, aortic atherosclerotic lesions werelarger compared to ApoE KO mice despite no significant differ-ences in plasma cholesterol or in the distribution of lipoprotein

86 G. Tibolla et al. / Atherosclerosis 210 (2010) 78–87

Fig. 7. Relative expression of inflammatory mediators and of their receptors in the vascular wall measured by quantitative real-time PCR. Analysis of adhesion moleculese on (Apa rmalie crogram (figur

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xpression in APP23/ApoE KO and ApoE KO mice. Panel A: Relative mRNA expressind the adhesion molecules ICAM-1 and VCAM-1 (n = 14 for each group). Results, noxpressed as mean ± S.E., *p < 0.05 vs. ApoE KO). Panel B–I: representative photomiice (B, C, F and G) and from APP23/ApoE KO mice (D, E, H and I). Scale bar: 10 �m

articles were observed between the two groups. Furthermoretherosclerotic plaques from APP23/ApoE KO mice showed anncreased presence of macrophages. This effect was associated withncreased expression of MCP-1 and IL-6, two key players in theathogenesis of cardiovascular diseases [27], as well as that of

CAM-1.Although young APP23/ApoE KO fed an atherogenic diet show

ncreased A� peptide levels in the brain, A� neither is depositedn cerebral tissues nor causes a massive neuroinflammation, but its associated with mild activation of macrophages and endothelialells in the brain. The endothelial dysfunction can also be appre-iated in the peripheral vasculature: indeed, aortic and coronaryndothelial cells, particularly susceptible to stress induced hyper-holesterolemia, showed increased levels of ICAM-1 compared topoE KO mice, despite the absence of A� peptides in plasma as wells in the atherosclerotic plaque.

Oxidative stress has been suggested to be one of the earliestathological changes in the brain of Alzheimer disease patients28]. The absence of a relevant oxidative stress in the brain of

PP23/ApoE KO mice supports our observation that vascular acti-ation precedes neuronal alteration in these mice and, as previouslyeported by several authors [20,21,29], it is probably associatedith a direct effect of soluble aggregates of A� peptides, on brain

ndothelial dysfunction.

oE KO = 1) of MCP-1, IL-6, IL-10, MIP-1�, the chemokine receptors CCR1 and CCR2zed for the RLP13a expression, are shown as fold of induction vs. ApoE KO (data arephs of ICAM-1 expression in aortae (B–E) and coronary artery (F–I) from ApoE KOe B–I).

In conclusion the present study documents, for the first time,that neuronal overexpression of A� in atherosclerosis-prone miceresults in activation of macrophage cells and endothelial dysfunc-tion, which promote atherogenesis. Our work confirms and extendsthe findings by Li et al. [9] since it demonstrates that cerebrallocalised A� overexpression sustains the inflammatory responseand the progression of atherosclerosis in a mouse model, by devel-oping endothelial dysfunction and atherosclerosis. Of note, Puglielliet al. [7] showed that A� catalyses 4-cholesten3-one formation andsuggested that this mechanism may be responsible for increasedatherogenesis in Tg2576 animals. In our animal model we did notobserve differences in 4-cholesten-3-one concentrations thus sug-gesting that additional mechanisms, yet unknown, are responsiblefor the link between APP overexpression to inflammation, oxidativestress and increased atherosclerosis.

Nevertheless, the convergence of risk factors for AD andatherosclerosis suggests that these chronic inflammatory diseasesmay have overlapping pathogenetic mechanisms in which choles-terol, lipid peroxidation and A� peptides play a central role. Finally,

as the pharmacological neutralization of the ApoE/A� interactionin the brain is currently under intensive investigation as a potentialtherapeutic strategy [30], our data suggest that, when the ApoE/A�interaction is completely abolished following ablation of ApoE, pre-cautions should be considered as the increased clearance of A�

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eptides from brain, could on the other hand, promote peripheraleleterious effects finally resulting in increased atherosclerosis.

cknowledgements

This work was supported by grants from PRIN 2004 and PRIN006, SISA (Società Italiana Studio Aterosclerosi sezione Lombar-ia), Ministero dell’Istruzione, Università e della Ricerca (MIUR) (to.L.C.) and the European projects: DIMI (LSHB-CT-2005-52146 to.M.) and EWA (LSHM-CT-2005-518245 to A.M. and E.V.).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.atherosclerosis.2009.10.040.

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