UvA-DARE (Digital Academic Repository) …A magnetic resonance angiogram was made as an anatomical reference. A C57Bl/6J mouse was anaesthetized with isoflurane 1-2% and imaged using

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Neuropathological changes in mouse models of cardiovascular diseases

Bink, D.I.

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Citation for published version (APA):Bink, D. I. (2016). Neuropathological changes in mouse models of cardiovascular diseases.

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3 |Lack of intracranial atherosclerosis in various atherosclerotic mouse models

Diewertje I Bink†, Katja Ritz†, Claire Mackaaij, Mark R Mizee, Hanneke J Ploegmakers, Onno J de Boer, Judith C Sluimer, Guido RY De Meyer, Louise van der Weerd, Helga E de Vries, Mat JAP Daemen

† Diewertje I Bink and Katja Ritz are co-first authors

Manuscript in preparation

Bink DB Proefschrift 160218.indd 75 10-05-16 16:35

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AbstractAims Although mice are used extensively to study atherosclerosis of different vascular beds, limited data is published on intracranial atherosclerosis. Since intracranial atherosclerosis is a common cause of stroke and is associated with dementia, a relevant animal model is needed to study these diseases. Here, we examined the presence of intracranial atherosclerosis in different atherogenic mouse strains and studied differences in vessel wall characteristics in search for possible explanations for the different atherosclerotic susceptibility between extracranial and intracranial vessels.

Method and results The presence of atherosclerotic plaques was systematically examined from the distal portion of the common carotids to the circle of Willis in three atherogenic mouse models: ApoE-/-, ApoE-/-Fbn1C1039G+/- and ApoB100/LDLr-/-. Extra- and intracranial vessel characteristics were studied by immunohistochemistry. All three strains developed atherosclerotic lesions in the common carotids, while no lesions were found intracranially. Atherosclerotic plaques were frequently present in the internal carotids, but usually stopped at the bifurcation with the pterygopalatine artery, which coincided with altered vessel morphology. Intracranially, the number of elastic layers decreased, the internal elastic lamina became thicker, an endothelial cell activation marker decreased, and tight junction marker claudin-5 increased. Stimulation of brain endothelial cells with oxLDL induced endogenous protective antioxidant capacity through a Nrf2-mediated increase of heme oxygenase-1 expression.

Conclusions Intracranial atherosclerosis is absent in all three atherogenic mouse models up to the age of 41 weeks. We suggest that differences in brain vessel structure and increased antioxidant capacity of the brain endothelium contribute to decreased atherosclerosis susceptibility of murine intracranial arteries.


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Lack of intracranial atherosclerosis in mice

IntroductionIntracranial atherosclerosis is the cause of at least 10% of ischemic strokes and is associated with dementia.1-3 The prevalence of intracranial atherosclerosis varies per ethnicity and increases from about 23% of the population at the age of 50-59 to 80% at the age of 80-89.4 It becomes increasingly clear that distinct vessel and atherosclerotic plaque characteristics of intra- and extracranial arteries occur in humans.5 Intracranial atherosclerosis develops approximately 20 years later compared to coronary or carotid atherosclerosis and shows a more stable and less severe phenotype than extracranial atherosclerosis. The underlying mechanisms of these striking differences are largely unknown, but may be related to a specific constitution of intracranial vessels as observed in different species.5 For example, intracranial vessels have fewer elastic fibers than extracranial vessels, a distinct vessel wall metabolism and glycocalyx composition, elevated endogenous antioxidant enzyme activity and exhibit tight junctions between the endothelial cells lining the vessel wall.5-7 In addition, a reduction in vessel wall permeability and lipoprotein deposition in the vessel wall of intracranial arteries compared to extracranial arteries have been observed in monkeys and rabbits.6, 7

Only few studies have been published on the occurrence and frequency of atherosclerotic plaques within the skull or brain of experimental animals. Large animals such as dogs, monkeys and swine develop large visible atherosclerotic lesions.8-10 However, small-sized animals, like rats and rabbits, only develop small lesions with intimal thickening and foam cell formation in the intracranial vessel wall.11-13 Studies on atherosclerosis in murine intracranial vessels have barely been reported. The presence of intracranial atherosclerosis in the circle of Willis (CoW) has been stated in the crossbred of LDLr-/- with hApoB expression on chow diet, however no quantitative data on plaque size or phenotype were shown.14 The absence of atherosclerosis in the CoW was reported in 80 weeks old ApoE-/-LDLr-/- mice and 18 months old ApoE-/- mice on Western-type diet (WD).15, 16 In addition, absence of atherosclerosis has been reported in ApoE-/- mice in the middle cerebral artery (MCA) and basilar artery (BA) at the age of 29 weeks, in the cerebral arterioles at the age of 7-13 months on WD and in cerebral microvessels at the age of 8-10 months on chow, although in the latter lipid laden macrophages were observed in the perivascular region.17-19

To determine the presence or absence of intracranial atherosclerosis in severe atherosclerotic mice, we collected the heads of three atherosclerotic mouse models (ApoE-/- mice, ApoE-/-Fbn1C1039G+/-, and ApoB100/LDLr-/- mice) that had already been used in other studies. Intracranial vessels were systematically analyzed for the


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presence of atherosclerosis. Moreover the precise transition point from atherosclerotic to non-atherosclerotic vessels area was determined. To determine plausible causes of the reduced susceptibility of intra- and extracranial arteries we studied structural intra- and extracranial vessel wall characteristics in C57Bl/6 mice and the response of cultured brain endothelial cells to oxidized LDL (oxLDL), mimicking pro-atherosclerotic conditions.

Materials and MethodsAnimalsThirty-three mice of three genetically modified mouse models were obtained from different laboratories to study the presence of atherosclerosis. Four ApoE-/- on a chow diet at the age of 50-52 weeks (males and females), eight ApoE-/- on Western-type diet (WD) at the age of 28-41 weeks (males and females), twelve ApoE-/-Fbn1C1039G+/- on WD at the age of 21-41 weeks (females) and eight ApoB100/LDLr-/- mice (males and females) ranging from 37-41 weeks of age (also shown in table 1). ApoE-/- and ApoE-/-Fbn1C1039G+/- were fed a WD at the age of 6 weeks, hApoB100/LDLr-/- female mice at the age of 12 weeks and ApoB100/LDLr-/- male mice the age of 32 weeks. Food and water were available ad libitum. For all ApoE-/- and ApoE-/-Fbn1C1039G+/- mice the complete head and neck including the vascular tree were retrospectively harvested from other experimental studies and complemented with ApoB100/LDLr-/- mice, leading to differences between the experimental groups in age and diet.20 Differences in ages additionally occurred due to premature death. All studies were approved by local ethical committees and were performed in accordance with national and European regulations.

MRIA magnetic resonance angiogram was made as an anatomical reference. A C57Bl/6J mouse was anaesthetized with isoflurane 1-2% and imaged using a horizontal 7T magnet (Pharmascan, Bruker, Rheinstetten, Germany) with a circular polarized MRI transceiver coil for 1H with an inner diameter of 23 mm. Bruker Paravision 5.1 software was used for image acquisition. A T2 3D Flash sequence was used to image linear flow in the mouse neck and head with the following parameters: TR 15 ms, TE 2.6 ms, NA 4, matrix 256x256x256, FOV 17x17x20 mm, FA 30º, resolution 66x66x78 µm/pixel, scan time 36 min. The vasculature segmentation was done with AMIRA (version 9.0).


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CTA CT scan was made for a three-dimensional overview of the vascular tree and its entry into the skull. A mouse head was perfused with MicroFil (Flow Tech, Carver, MA, USA). A CT scanner (Skyscan 1076, Bruker, Rheinstetten, Germany) connected to a camera (Princeton Instruments, Trenton, USA) was used to image the vasculature with the following parameters: Alu 0.5 mm filter, exposure time 1750 ms, voltage 48 kV, current 200 µA. Reconstruction was done with NRecon (version 1.5.1.4), resulting in an image of 1780x1760 pixels. Empty edges of the pictures were cropped with Photoshop (Adobe Photoshop CS6). Vasculature and bone segmentation was done with AMIRA (version 9.0).

Histology and immunohistochemistryMice were sacrificed by transcardial perfusion with PBS and 4% PFA after Pentobarbital injection or by CO2 inhalation. The heads were post-fixed in 4% PFA and placed for 3-4 weeks in 12.5% EDTA for decalcification. The skin was dissected from the skull and the heads were embedded in paraffin. Sections of atherogenic mice were cut at 10 µm thickness and every fifth section was stained with haematoxylin and eosin (HE) to assess the distribution of atherosclerotic lesions, ranging from foam cell detection in the vessel wall to advanced atherosclerotic plaques as defined by the American Heart Association (AHA) classification.21, 22 Lesions at the common carotid artery (CCA) bifurcation were classified according to the AHA classification. Six C57Bl/6 mouse skulls were decalcified and cut transversely at 5 µm thickness for a more detailed immunohistochemical analysis of vessel characteristics and morphology. HE stains were used to determine seven areas of interest (see figure 1B): just below the CCA bifurcation (nr. 1), internal carotid artery (ICA) before the bifurcation with the pterygopalatine artery (Ptgpal) (nr. 2), ICA after the bifurcation with the Ptgpal (nr. 3), ICA just below the skull base (nr. 4), ICA within the skull base (nr. 5), ICA just above the skull base (nr. 6), ICA at the start of circle of Willis (CoW) before the bifurcations to the posterior cerebral artery (PCA) and middle cerebral artery (MCA) (nr. 7). Adjacent sections were used for Elastica van Gieson (EvG) staining and immunohistochemistry. Immunohistochemistry was done with the antibodies listed in supplemental table 1.

Image analysisFrom the CCA and ICA immunohistochemistry sections pictures at 20x magnification were taken using a Leica DM5000B microscope equipped with a DFC500 camera


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(Leica, Germany) and LAS v4.5 software. Image analysis was performed with Image Pro Premier 9 (Media Cybernetics, Rockville, USA) to determine the optical density (OD) for PermaBlue stained IHC and the percent positive area. The vessel wall intima and media were selected as regions of interest for all stainings and vessels. Regions of interest of right and left arteries were averaged. Areas with folding and other artefacts were excluded from analysis. Vessel wall thickness was measured on the HE stained sections.

Cell culture and treatmentsThe immortalized human brain endothelial cell line hCMEC/D3 was cultured as described previously23, 24 and used at passages 27-33. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as described elsewhere25 and used between passages 2 and 3.Cells were treated with human oxLDL (BT-910, Biomedical technologies inc.) with TBARS ranging from 26.6 to 41.9 nmol of MDA/mg protein (Alfa Aesar, Ward Hill, MA, USA). Uptake of the oxLDL was confirmed with Dil-oxLDL (BT-920, Alfa Aesar, Ward Hill, MA, USA) and FACS analysis, quenching for surface fluorescence with Trypan blue.

RNA isolation and real-time quantitative PCRhCMEC/D3 cells were stimulated for 6 h and 24 h with 50 µg/ml and 100 µg/ml oxLDL (n=4), harvested using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) followed by the RNA isolation procedure following manufacturer’s guidelines. cDNA synthesis was performed with 1 µg RNA using the high capacity cDNA reverse transcription kit following manufacturer’s guidelines (Applied Biosystems, Foster City, CA, USA). cDNA was amplified using SYBR Green PCR Master Mix (Applied Biosystems) in a final volume of 10 µl on a ViiaTM 7 Real-Time PCR System (Applied Biosystems) using the following heme oxygenase-1 (HO-1) primers: HO-1 forward 5’-TGGAGCGTCCGCAAC-3’ and reverse 5’-TCCTTCAGGGTCTCTGACAA-3’. Expression levels were normalized to beta-2-microglobulin (forward 5’-GTATGCCTGCCGTGTGAAC-3’and reverse 5’-AAAGCAAGCAAGCAGAATTTGG-3’) and the 2-ΔΔCT method was used for data analysis.26

Western blotting and nuclear fractioningTo determine HO-1 protein levels using western blotting, hCMEC/D3 cells were cultured in 6 well microplates and stimulated for 24 h with oxLDL (n=3). Cells were


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lysed in cell lysis buffer (Cell Signaling Technology Inc, Boston, MA, USA) containing complete protease inhibitor cocktail (Roche, Almere, The Netherlands). Lysates were taken up in SDS sample buffer (100 mM Tris-HCL pH 6.8, 4% SDS, 20% glycerol, 5% β-mercaptoethanol) and heated to 95⁰C for 5 min. Lysates were resolved on a 10% SDS-polyacrylamide gel electrophoresis, blotted to PVDF membranes (Bio-Rad Laboratories, Berkeley, CA, USA) and incubated over night at 4⁰C with the primary antibodies rabbit-αHO-1 (1:1000, Enzo Life Sciences, Farmingdale, NY, USA) and goat-αactin (for normalization, 1:400; Santa Cruz Biotechnology) in Odyssey blocking buffer (LI-COR, Lincoln, AK, USA) diluted 1:1 in PBS and after blocking with Odyssey blocking buffer for 1 h at RT. Respective IRDye infrared fluorescent dyes secondary antibodies and the Odyssey infrared imaging system (Li-COR) were used for visualization and quantification of protein levels.To determine Nrf2 nuclear protein levels, hCMEC/D3 cells were cultured in 6 well microplates (2 wells per condition) and stimulated for 4 h with oxLDL (n=3). Cells were washed with ice-cold PBS and lysed and fractioned using the NE-PER nuclear and cytoplasmic extraction kit following the manufacturer’s instructions (Thermo Scientific, Rockford, IL, USA). Western blotting was performed as described above and using the primary antibodies rabbit-αNrf2 (1:400, Santa Cruz Biotechnology), goat-αlamin B (1:200, to normalize nuclear fraction; Santa Cruz Biotechnology) and goat-αactin (1:400, to normalize cytoplasmic fraction; Santa Cruz Biotechnology). Quantification of protein bands was performed using Odyssey imaging software. Fluorescence intensity was measured in an area surrounding the protein bands, and subtracted by the same area without bands for lane background correction. Relative Nrf2 protein levels were obtained by correcting for loading control (actin or lamin B) levels in each sample.

ROS measurement using flow cytometric analysishCMEC/D3 and HUVECs were cultured in 24 well plates and stimulated with 50 µg/ml and 100 µg/ml oxLDL for 24 h. Cells were washed and incubated at 37°C for 1 h with the intracellular ROS detection probe CM-H2DCFDA (Invitrogen). Endogenous ROS production was measured by measuring the median fluorescence intensity of 10.000 viable cells using a FACSCalibur flow cytometer (Becton & Dickinson, San Jose, CA, USA). The effects of oxLDL on endothelial ROS production was analyzed in both unstimulated and immune-activated cells, treated with a combination of TNF-α/ IFNγ (5 ng/ml each, 24 h).


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Statistical analysisData was analyzed using Graphpad Prism software (v5.01, La Jolla, CA, USA). Immunohistochemical measures were considered statistical significant if p<0.05 by Friedman test and posthoc tests were done with Dunns test to compare all pairs of columns. In vitro results are shown as median ± standard deviation for FACS results and as means ± standard deviation for others. Statistical significance was considered if p<0.05 by two-tailed Student’s t-test.

ResultsMapping of the mouse arterial organizationHistological and immunohistochemical stainings of transversal mouse head sections were studied to detect the distribution of atherosclerotic plaques and the vessel wall characteristics. For the nomenclature of vessels the rat anatomy atlas from Greene and the MRI results from Kara et al. were used.27, 28 For the assessment of atherosclerotic lesions we focused on the CCA, proximal parts of the external carotid artery (ECA), ICA, Ptgpal, CoW, vertebral artery (VA) and the BA (figure 1).

Figure 1. Three-dimensional reconstruction of the peripheral and cerebral arterial tree. A, CT scan showing a slightly ventrolateral view of the skull and vessels. The dashed line indicates where the atherosclerotic plaques stop in most mice. The continuous line indicates the place where the ICA enters the skull. Red indicates part of the vessels without atherosclerosis. The black indicates the part of the vessels with extensive atherosclerosis (based on 32 mice). The gray indicates atherosclerosis as observed only in one mouse. B, Magnetic resonance angiogram showing the sagittal view of the mouse arterial tree from common carotid artery (CCA) to the circle of Willis (CoW) and its main branches. The numbered blue areas indicate the areas in which immunohistochemistry sections were taken.


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Atherosclerotic lesions are absent in the craniumAll 32 animals developed atherosclerotic lesions in at least one of the CCAs, while intracranial atherosclerotic lesions were completely absent. Atherosclerotic lesions were frequently present in the ICA cranial from the bifurcation with the CCA, but typically stopped at the bifurcation with the Ptgpal, indicating a first transition point in atherosclerotic susceptibility (figure 1A and 2). Only three ApoE-/-Fbn1C1039G+/- mice out of all 32 mice showed early or advanced atherosclerotic lesions in the ICA after the ICA bifurcation with the Ptgpal; one of these showed lesions within the carotid foramen. However, there were no lesions in the vessels when they entered the cranial cavity, indicating a second transition point in atherosclerotic susceptibility between the extracranial and intracranial part of the ICA. In six mice lesions were observed in the extracranial portion of the VA, but not in the intracranial part nor in the BA (table 1).

Figure 2. Representative picture of the first transition point of the internal carotid artery (ICA) at the bifurcation (bif) with the pterygopalatine artery (Ptgpal) where the atherosclerotic lesion usually stopped in a 26 week old ApoE-/-Fbn1C1039G+/- mouse on WD. An atherosclerotic plaque containing a necrotic core with foamy macrophages is shown in the ICA on the rostral site of the bifurcation. There was no plaque at the cranial site of the bifurcation, the site of the vessel that is heading towards brain. In the left panel (2.5x) an overview is shown of the bifurcation of the ICA and Ptgpal, while the right panel (10x) shows the first transition point in more detail.


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BA: Basilar artery; CCA: Common carotid artery; CoW: Circle of Willis; ECA: External carotid artery; F: female; ICA: Internal carotid artery; M: male; Ptgpal: Pterygopalatine artery; VA: Vertebral artery; WD: Western-type diet.

Table 1. Presence of atherosclerotic lesions in individual mice of the three investigated atherogenic mouse strains


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Thus, none of the 32 mice showed intracranial atherosclerotic lesions, despite the advanced age of the mice, and the added burden of a WD in many of the studied groups.The severity of the atherosclerotic lesions was classified at the CCA bifurcation.21, 22 Almost all mice showed predominantly advanced lesions type V according to the AHA classification: the lesions contained large necrotic cores, cholesterol crystals, layers of fibrous connective tissue and macrophage foam cells mostly located in thin fibrous caps. Only one of the male ApoB100/LDLr-/- mice showed less advanced lesions (type III).Although an older age may have increased the atherosclerotic burden, later time points were not achievable in the ApoB100/LDLr-/- mice on long-term WD as they needed to be euthanized at 8.5 months of age because of large subcutaneous fat depositions in the paws and consequential muscle compression (data not shown). Numerous ApoB100/LDLr-/- (n=7 out of 8), ApoE-/-Fbn1C1039G+/- (n=11 out of 12) and ApoE-/- (n=3 out of 6) mice on WD had small to large xanthomas in their brains, mainly in and around the choroid plexus and neocortex, as shown by Van der Donckt et al.29 Furthermore, ApoB100/LDLr-/- (n=7 out of 8), ApoE-/-Fbn1C1039G+/- (n=10 out of 12) and ApoE-/- (n=4 out of 6) mice on WD and ApoE-/- (n=2 out of 4) mice on chow had cholesterol granulomas in their middle and inner ear affecting surrounding tissue and blocking the perception of auditory signals.

Intra- and extracranial vessel wall characteristics differNext, we looked for vessel wall characteristics in the intima and media that may be linked to the profound difference in the susceptibility of atherosclerosis development between extracranial and intracranial arteries. Several changes in vessel wall characteristics were observed. Examples of the CCA, the extracranial ICA and the intracranial ICA are shown in figure 3. Numbers in the figure and text indicate the areas where the sections for immunohistochemistry were taken and are shown in figure 1B. The vessel wall thickness decreased from the CCA towards the intracranial part of the ICA (59±9 µm vs 29±5 µm, p=0.0003) (figure 3A-D). The CCA (nr. 1 in figure 1B) and ICA before the bifurcation with the Ptgpal (nr. 2) had 3-4 elastica lamina layers. Beyond the Ptgpal bifurcation (nr. 3) the ICA contained only 2 elastica lamina layers. The intracranial part of the ICA showed a thick IEL and a discontinuous elastic layer in the media (nr. 6 and 7) (figure 3E-H).The relative amount of smooth muscle cells (SMCs) in the vessel wall and the differentiation level of the SMCs were measured with smooth muscle actin (SMA) and smooth muscle myosin heavy chain (SM-MHC), respectively. The percent positive


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Figure 3. Changes in vessel wall morphology in C57Bl/6 mice from the common carotid artery (CCA) to the intracranial part of the internal carotid artery (ICA) (n=6). A-D, Vessel wall thickness of the intima and media was measured in the HE staining. Number of elastic fibers in the Elastica van Gieson (EvG) staining (E-H) and Icam-1 positive staining (M-P) decreased, while the amount of SMA (I-L), claudin-5 (Q-T), HO-1 (U-X) and NQO1 (Y-AB) increased from the CCA bifurcation site to the intracranial part of the ICA (63x magnification). Numbers on the x-axis indicate the position in the vessels as shown in figure 1B. Dashed lines indicate the first transition point of the ICA bifurcation with the Ptgpal where atherosclerosis stopped in most animals. Continuous lines indicate the second transition point and entry of the ICA in the skull. Statistical significance was determined by Friedman test and considered statistical significant if p<0.05.


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area and intensity of SMA increased intracranially, indicating a relative increase of SMCs in the vessel wall (OD and percent area p<0.0001; figure 3I-L). This increase of SMCs in the vessel wall could be due to the decrease in elastin fibers, indicating the transformation from an elastic artery to a more muscular artery. There was a similar increase in the expression of SM-MHC (differentiated amount of SMCs) compared to the SMA (total amount of SMCs) between extra- and intracranial parts of the ICA, resulting in an unchanged smooth muscle differentiation percentage intracranially compared to extracranially (OD p=0.3356, percent area p=0.0724).

The adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) establish the adhesion of monocytes to endothelial cells and thereby facilitate atherogenesis.30, 31 ICAM-1 and VCAM-1 were present in endothelial cells in all examined vessels. ICAM-1 staining, however, became less abundant as the ICA enters the skull (OD p<0.0001, percent area p=0.0161; figure 3M-P). Posthoc differences in ICAM-1 intensity were significant between CCA (nr. 1) and extracranial parts of the ICA (nr. 2 and 4) compared to the intracranial part of the ICA (nr. 7 and 6 compared to nr. 1 in figure 1B). The intensity of the VCAM-1 staining did not significantly differ between measurement points within the vessel (p=0.0620). There was a significant difference in VCAM-1 positive percent area between measurement points (p=0.0006), but there was no linear trend from extracranial to intracranial arteries or the other way around (data not shown).In order to investigate potential changes in the hypoxia response pathway, we investigated the glucose transporter 1 (Glut-1) which is upregulated in response to hypoxia as a downstream target of HIF1α.32 Glut-1 staining was virtually absent in the CCA, ECA and extracranial ICA. There was a slight increase in the intracranial part of the ICA (percent area p=0.0313), but Glut-1 only became clearly positive at the bifurcations of the CoW and in the brain capillaries (data not shown).Caveolin is an integral membrane protein that is involved in endocytosis and acts proatherogenic.33, 34 Although there was a significant difference in caveolin intensity throughout the segments as shown with the Friedman test (OD p=0.0226, percent area p=0.0491), there was no linear trend from extracranial to intracranial arteries or the other way around (data not shown).To investigate paracellular permeability through the intima, the expression of tight and adherent junction molecules, like claudin-5, ZO-1 and VE-cadherin were examined. Claudin-5 stained only weakly in the CCA and ECA, but increased substantially passing the skull base (OD and percent area p<0.0001) (figure 3Q-T). Tight junction


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markers ZO-1 and adherent junction marker VE-cadherin did not show a difference in expression between the different points (ZO-1 OD p=0.2712, percent area p=0.0531; VE-cadherin OD p=0.8841, percent area p=0.2830).Oxidative stress is also known to be involved in atherosclerosis development.35,

36 Antioxidant enzyme expression was determined with HO-1 and NAD(P)H dehydrogenase, quinone 1 (NQO1) stainings. HO-1 is an endogenous antioxidant enzyme highly induced by oxLDL. The basal levels of HO-1 increased from CCA to the intracranial part of the ICA (OD p=0.0281, percent area p=0.0039; figure 3U-X). In addition, there was an increase in intracranial antioxidant enzyme NQO1 expression compared to extracranial expression (percent area p=0.0072) (figure 3Y-AB). Posthoc differences were significant between the ICA before the Ptgpal bifurcation (nr. 2) compared to the intracranial part of the ICA (nr. 6 and nr. 7).Expression of the different markers followed similar patterns in two 18 week old ApoE-/- mice on chow (data not shown).

OxLDL induces protective antioxidant pathways via Nrf2 translocation in brain endothelial cellsBased on our immunohistochemical data, we postulate that enhanced levels of anti-oxidant enzymes protect the intracranial vasculature from atherosclerotic plaque development. To elucidate if the antioxidant response is upregulated in human brain endothelial cells under pro-atherosclerotic conditions, brain endothelial cells were stimulated with oxLDL. Interestingly, upon the treatment of the endothelial cells with oxLDL, we observed a significant induction of HO-1 on mRNA and protein level (p<0.0001; figure 4A-B). Moreover, oxLDL induced the translocation of the transcription factor nuclear factor (erythroid-derived-2)-like (Nrf-2) a key regulator of the endogenous antioxidant enzymes, including HO-1, to the nucleus of the brain endothelium 4 h after stimulation with oxLDL (p=0.0249; figure 4C). Treatment of brain endothelial cells with oxLDL significantly protected brain endothelial cells from oxidative stress as indicated by a reduced production of ROS in both untreated cells and cells treated with the pro-inflammatory cytokines TNF-α/ IFNγ (100 µg/ml: oxLDL untreated cells p=0.0024; treated cells p<0.0001; figure 4D). In contrast, this protective effect was not observed in HUVECs upon similar conditions (figure 4D).


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DiscussionThis study demonstrates that genetically modified mice with moderate or severe extracranial atherosclerosis do not develop intracranial atherosclerosis until at least the age of 41 weeks. Our results also indicate a first transition point in atherosclerotic susceptibility at the bifurcation of the ICA and the Ptgpal after which no atherosclerosis develops in most mice and a second transition point at the entry of the cranium after which no atherosclerosis was observed in all the mice studied. At these

Figure 4. oxLDL protects brain endothelial (hCMEC/D3) cells from oxidative stress. A, HO-1 expression levels are increased after 6 and 24 h (n=4). B, Quantification of HO-1 protein expression as measured by western blot, corrected for actin (n=3). C, Western blot analysis of nuclear and cytoplasmic fractions of brain endothelial cells corrected for laminB and β-actin, respectively, showed an increased nuclear presence of Nrf2 (n=4). D, oxLDL significantly reduced brain endothelial reactive oxygen species (ROS) production in untreated and immune-activated (TNF-α/IFNγ) conditions (n=3). Expression data are presented as means ± SD. ROS production is presented as median fluorescent intensity (MFI) percentage compared to control ± SD. Statistical significance was determined by two-tailed Student’s t-test. *p<0.05, **p<0.001, ***p<0.0001.


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transition points we report vessel wall changes such as reduced elastin layers, a reduced endothelial activation, an increase in tight junction protein claudin-5 and an increased expression of the protective antioxidant enzymes NQO1 and HO-1, which may be associated with the observed atheroprotected phenotype of intracranial arteries. We also show that brain endothelial cells are protected against inflammation-induced oxidative stress by a pathway activated by oxLDL and also suggest a role for Nrf2-mediated HO-1 expression in this response.

Unlike extracranial atherosclerosis, studies on intracranial atherosclerosis are barely reported in mice. To the best of our knowledge, we are the first group systematically studying the presence of atherosclerosis throughout the ICA in multiple atherogenic mouse models. In ApoE-/- mice of 9 months on chow or high-fat diet stenotic lesions have been reported in the ECA and ICA.37, 38 The authors however only studied the proximal part of the ICA, since the dissection of the distal ICA is difficult due its location between bone structures. We solved this technical problem by 1) decalcifying the entire skull in EDTA for 3-4 weeks and 2) sectioning the entire decalcified skull. Across the dozens of atherosclerotic mouse models available, only four other studies have been published on intracranial atherosclerosis in the CoW or its main branches. In genetically modified ApoE-/-LDLr-/- mice of 80 weeks of age on WD no atherosclerotic lesions were found in the CoW.15 Intracranial atherosclerosis was also absent in ApoE-/- mice on WD at the age of 29 weeks or at the age 18 months fed with WD for 6 months.16, 19 Atherosclerosis in the CoW has been reported in male LDLr-/-:hApoB+/+ mice of 12 months on chow diet.14 Unfortunately, the actual data is not presented in that paper and lesion type is not mentioned. We therefore also included male ApoB100/LDLr-/- mice in our study. The absence of intracranial atherosclerosis in the 9 months old ApoB100/LDLr-/- mice on a high-fat diet could be explained by the younger age of our mice and the difference in ApoB subtype. We however expect that the high-fat diet would increase the amount of atherosclerosis leading to an earlier onset of possible intracranial atherosclerosis. Unfortunately, we could not investigate the presence at later time points due to harmful cerebral, inner ear and subcutaneous fat depositions which occurred without or prior to fat depositions in intracranial vessels.

Our results suggest that the difference in atherosclerotic susceptibility between extra- and intracranial vessels may be caused by the endothelial heterogeneity. Brain capillary endothelial cells are highly specialized and are part of the blood-brain barrier (BBB), which has a low permeability to protect the brain from toxic substances, accomplished


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by the high expression of tight junction markers and selective transporters. Brain-related factors relevant for differentiating endothelial cells into the BBB phenotype may also influence the morphology and reactivity of larger intracranial arteries, as we found an increase in markers important for the BBB function, like claudin-5 and Glut-1 in the large arteries in the skull compared to the extracranial part of the ICA. This gradual increase can give rise to reduced vessel wall permeability and protect the intracranial part of the ICA from atherosclerosis development.Only few animal studies investigated differences between extra- and intracranial vessels. In rabbits it is shown that the SMC mediated sensitivity to l-norepinephrine differs in different parts of the ICA, marked by an abrupt transition point before its entry through the skull.39 Concanavalin A, which reacts with certain glycocalyx molecules, did not react with the cerebral vessels of rabbits and monkeys, but did with the aorta, coronary, femoral and carotid arteries.40 Differences in vascular response between intra- and extracranial vessels to compounds are probably due to differences in vessel wall characteristics. Pro-atherosclerotic compounds like oxLDL stimulate ICAM-1 and VCAM-1 expression and thereby increase monocyte adhesion and plaque formation.41, 42 In rabbits oxLDL particles impaired the contraction and relaxation of carotid arteries, but not of the basilar artery.43 Differences in oxLDL response between extra- and intracranial vessels have also been shown in quails. Interestingly, brain microvascular endothelial cells had a lower death rate in response to oxLDL than carotid endothelial cells.44 This was accompanied by a higher response of HO-1 mRNA and enzyme activity, which is in line with our findings in the human endothelial cells. A better antioxidant defense has also been shown in human intracranial arteries.45 We could further link the increased HO-1 mRNA and protein expression in the microvascular endothelial cells to the activation of Nrf2, a key player for the regulation of genes involved in vascular antioxidant defense pathways. Also, cytokine-induced oxidative stress was significantly reduced in oxLDL-stimulated brain endothelial cells but not in HUVECs, suggesting the induction of an oxLDL-mediated protective response specific to brain endothelial cells. HO-1 is known for its atheroprotective role and its induction has been associated with a reduced plaque formation in mice.46 An increased HO-1 expression has also been associated with a reduction of leukocyte adhesion and monocyte chemotaxis.47-49 The increased HO-1 expression that we observed in intracranial mouse arteries therefore suggests a reduced sensitivity to leukocyte adhesion and monocyte chemotaxis and is in line with the decreased expression of endothelial activation marker ICAM-1 intracranially. The coinciding increased NQO1 enzyme expression is also activated by Nrf2 and has


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likewise been associated with decreased atherosclerosis susceptibility.50, 51 The enhanced expression of tight junctions may further contribute to a reduction in vessel wall permeability, together leading to a reduced susceptibility to develop atherosclerosis. Although causality has not been shown yet, changes in elastic layers have also been suggested to influence atherosclerosis vulnerability.52

A limitation of this study is that the cell lines used in the in vitro study are derived from a different species compared to the species in the ex vivo studies. The in vitro results have to be reproduced in mouse endothelial cell lines to verify whether similar protective mechanisms apply in both species. In addition, we used an immortalized brain cell line (hCMEC/D3) and compared these results with the results of a primary cultured extracranial cell line (HUVECs). Primary cultures of CNS-derived endothelia are phenotypically unstable, undergo rapid cellular senescence and usually fail to develop functional tight junctions, which limits the usefulness of these cells as in vitro models of the BBB.24 To overcome these problems, the hCMEC/D3 cell line is transduced several-fold. Although the cells retain most of the functional and morphological characteristics of BBB cells, it cannot be excluded that the cells may react differently compared to primary cell lines or endothelial cells in vivo.To conclude, we report a lack of intracranial atherosclerosis in various atherogenic mouse models up to the age of 41 weeks. These atherogenic mouse models are therefore unsuitable for studying the effects of intracranial atherosclerosis on the development brain pathology and dementia. Differences in vessel morphology and increased antioxidant capacities may contribute to the decreased susceptibility for developing atherosclerotic lesions in intracranial arteries. In addition, we suggest that HO-1 and its regulation via the Nrf2 pathway may play a crucial role in this protective response. Despite the lack of intracranial atherosclerosis, atherogenic mouse models like ApoE-/- and ApoB100/LDLr-/- mice have shown presence of brain pathology like increased cerebral inflammation, increased BBB permeability, reduction of neurogenesis and synapses and cognitive impairment compared to C57Bl/6 mice before the age of 9 months.53 The observed brain pathology will therefore not be a direct effect of intracranial atherosclerosis on the surrounding tissue.

AcknowledgementsWe thank Dr. C. Van der Donckt and L. Roth of the University of Antwerp for providing ApoE-/- and ApoE-/-Fbn1C1039G+/- mouse tissue; Dr. P.H.A. Quax and Dr. M.R. de Vries of the LUMC for providing ApoB100/LDLr-/- mouse tissue. We thank E. Suidgeest and I. Que for their technical assistance.


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This work was supported by the Netherlands CardioVascular Research Initiative: ”the Dutch Heart Foundation, Dutch Federation of University Medical Centres, the Netherlands Organisation for Health Research and Development and the Royal Netherlands Academy of Sciences” (CVON 2012-06).

Conflict of InterestThe authors declare no conflict of interest.


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Supplementary tableSu

pple

men

tary

Tab

le 1

. Prim

ary

antib

odie

s, s

econ

dary

ste

ps a

nd c

hrom

ogen

s us

ed in

this

stu

dy

AP

: Alk

alin

e P

hosp

hata

se; C

itr: c

itrat

e; D

AB

: 3,3

′-Dia

min

oben

zidi

ne; G

lut-1

: Glu

cose

tran

spor

ter 1

; HO

-1: H

eme

oxyg

enas

e-1;

ICA

M-1

: In

terc

ellu

lar A

dhes

ion

Mol

ecul

e 1;

NQ

O1:

NA

D(P

)H d

ehyd

roge

nase

, qui

none

1; S

MA

: Sm

ooth

mus

cle

actin

; SM

-MH

C: S

moo

th m

uscl

e m

yosi

n he

avy

chai

n; T

E: T

ris-E

DTA

; VC

AM

: Vas

cula

r cel

l adh

esio

n pr

otei

n; V

E-c

adhe

rin: v

ascu

lar e

ndot

helia

l cad

herin

; ZO

-1: z

ona

occu

lden

s-1.


Documents

UvA-DARE (Digital Academic Repository) …A magnetic resonance angiogram was made as an anatomical reference. A C57Bl/6J mouse was anaesthetized with isoflurane 1-2% and imaged using