Attenuated atherogenesis in apolipoprotein E-deficient mice lacking amyloid precursor protein

Atherosclerosis 216 (2011) 54–58

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Attenuated atherogenesis in apolipoprotein E-deficient mice lacking amyloid precursor protein Tim J.L. Van De Parre, Pieter-Jan D.F. Guns, Paul Fransen, Wim Martinet, Hidde Bult, Arnold G. Herman, Guido R.Y. De Meyer ∗ Division of Pharmacology, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp (Wilrijk), Belgium

a r t i c l e

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Article history: Received 6 July 2010 Received in revised form 17 January 2011 Accepted 18 January 2011 Available online 26 January 2011 Keywords: Amyloid precursor protein Atherosclerosis Atherogenesis Smooth muscle cells

a b s t r a c t Objective: Recent evidence suggests that amyloid precursor protein (APP) is overexpressed in atherosclerosis-prone regions of mouse aorta. We therefore investigated in the present study whether APP has a role in the progression and composition of atherosclerotic plaques. Methods and results: Apolipoprotein E-deficient (apoE−/− ) mice were crossbred with animals lacking APP (APP−/− ). After 16 weeks on a Western-type diet, apoE−/− and APP−/− /apoE−/− mice showed similar cholesterol levels. However, atherosclerotic plaque size was significantly reduced in the distal thoracic aorta (90% reduction) and abdominal aorta (75% reduction) of APP−/− /apoE−/− mice as compared to apoE−/− . Plaques at the level of the aortic valves were not different in size, but showed a more stable phenotype in APP−/− /apoE−/− mice, as indicated by a reduced macrophage content, an increased amount of collagen and a thicker fibrous cap. Conclusion: Our findings provide evidence that lack of APP attenuates atherogenesis and leads to plaque stability. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Atherosclerosis develops at predilection sites in the vasculature and depends on both biochemical and biomechanical stimuli [1]. Recently, we compared gene expression profiles of smooth muscle cells (SMCs) in an atherosclerosis-prone (ascending aorta, aortic arch and proximal thoracic aorta) and atherosclerosisresistant region (central thoracic aorta) of young (4 months) apoE−/− mice, thus before plaque development [2]. Among the differentially expressed genes, a significant upregulation of amyloid precursor protein (APP) was found in atherosclerosis-prone regions of the thoracic aorta [2]. APP is an integral membrane protein widely expressed in cells throughout the body, but hitherto mainly studied in the pathology of Alzheimer’s disease (AD) [3]. Given that APP is overexpressed in atherosclerosis-prone regions of mouse aorta [2], it is plausible to assume that APP levels are associated with plaque development and thus, apart from epidemiological and pathophysiological factors, may serve as a biochemical link between atherosclerosis and AD [4]. One finding in favour of this theory is that apolipoprotein E-deficient (apoE−/− ) mice, overexpressing the Swedish mutated (K670N/M671L) human APP (APP23) selectively in the brain, develop larger and more

∗ Corresponding author. Tel.: +32 3 2652737; fax: +32 3 2652567. E-mail address: [email protected] (G.R.Y. De Meyer). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.01.032

inflamed atherosclerotic lesions as compared to apoE−/− mice [5]. In the present study, we crossbred apoE−/− mice with animals lacking APP (APP−/− ) to further investigate whether APP has a role in the progression and composition of atherosclerotic plaques. 2. Materials and methods 2.1. Animals APP−/− mice (gift of Merck Research Laboratories, Rahway, NJ) and apoE−/− mice (Charles River Laboratories, Wilmington, MA), both backcrossed in C57Bl/6 background for more than 10 generations, were crossbred to generate double knockout animals (APP−/− /apoE−/− ). Offspring was genotyped for APP and apoE deficiency by PCR analysis using standard procedures. For APP, the following primers were used: 5 -CTG CTG CAG GTG GCT CTG CA-3 (sense), 5 -CAG CTC TAT ACA AGC AAA CAA G-3 (antisense) and 5 -CCA TTG CTC AGC GGT GCT G-3 (antisense for the disrupted gene) For apoE, we used 5 -GGA TTA CCT GCG CTG GGT GC-3 (sense), 5 -TGG AAG ATC TCC GCC TGC AG-3 (antisense) and 5 TGG CGG ACC GCT ATC AGG AC-3 (antisense for the disrupted gene). The expected sizes of amplified fragments were 250 bp for wild-type APP allele, 470 bp for disrupted APP allele and 671 bp for wild-type and disrupted apoE allele. The latter fragments were identified in two separate PCR reactions. At the age of 5 weeks, male

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Table 1 Body weight, plasma lipid values and plaque characteristics in APP−/− , apoE−/− and APP−/− /apoE−/− mice after 16 weeks on a Western-type diet.

Body weight, g Total cholesterol, mmol/l Triglycerides, mmol/l Plaque area, 103 ␮m2 Aortic valves Aortic root Aortic arch TA segment 1 TA segment 2 TA segment 3 TA segment 4 TA segment 5 Abdominal aorta Plaque composition Mac-3 positive area (%) ␣-SMC actin positive area (%) Maximal thickness fibrous cap (␮m) Sirius red positive area (%) PCNA positive area (%) Cleaved caspase-3 positive area (%)

APP−/−

apoE−/−

APP−/− /apoE−/−

27.5 ± 1.2 4.0 ± 0.3 0.59 ± 0.07

38.4 ± 1.2*** 24.8 ± 2.3*** 2.47 ± 0.36***

26.0 ± 0.6††† 23.1 ± 1.2*** 1.35 ± 0.14**,

n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.

409 85 16 7 1.3 1.2 1.3 15.5 31

± ± ± ± ± ± ± ± ±

52 13 8 3 0.9 1.2 0.9 8.0 16

517 103 14 8 0.4 0 0.8 1.3 8

± ± ± ± ± ± ± ± ±

36 19 7 6 0.4 0 0.5 0.6† 3†

n.a. n.a. n.a. n.a. n.a. n.a.

38 2.9 73 33 2.2 3.0

± ± ± ± ± ±

3 0.5 6 2 0.9 0.3

24 3.7 104 46 0.4 2.9

± ± ± ± ± ±

2†† 0.5 6†† 2††† 0.2† 0.4

††

TA: Thoracic aorta. n.a.: not applicable (no plaques). Data are presented as mean ± SEM. n = 17 for APP−/− , n = 10–14 for apoE−/− and n = 20–23 for APP−/− /apoE−/− mice. **P < 0.01, ***P < 0.001 as compared to APP−/− ; † P < 0.05, †† P < 0.01, ††† P < 0.001 as compared to apoE−/− . For body weight and plasma lipid values, ANOVA was followed by the Bonferroni test. For plaque area and composition, the unpaired Student’s t-test was used. If the variances were not homogenous, data were logarithmically transformed.

mice were fed a Western-type diet (Harlan Teklad, TD88137) for 16 or 32 weeks. We used male mice in the present study because at atherosclerosis-prone locations (before actual lesion development) upregulation of APP is more pronounced in male apoE−/− mice [2]. Blood samples were collected from 21-week-old mice via the retro-orbital plexus and were collected into EDTA tubes. Plasma was obtained by centrifugation (3 min, 1000 × g). Total cholesterol and triglycerides were determined using commercial enzymatic assays (Randox, Crumlin, UK). A limitation of the present study is that we do not know the plasma levels of low density lipoprotein (LDL) cholesterol and high density lipoprotein (HDL) cholesterol. The study was approved by the Ethical Committee of the University of Antwerp.

2.2. Histology and morphometry After anesthesia (sodium pentobarbital, 75 mg/kg, i.p.), the aorta was carefully removed and systematically divided into different segments: the aortic arch, thoracic aorta (TA) segments 1 through 5, and the abdominal aorta (Supplementary Fig. 1). Each segment was fixed in 4% formalin for 24 h, paraffin embedded and sectioned. Besides the different aortic segments, the heart was removed to analyze plaques at the aortic valves. Sections were stained with hematoxylin/eosin for general histological evaluation. Sirius red was used for collagen detection and Verhoeff’s elastic stain for the measurement of the plaque area. For immunohistochemistry, the following primary antibodies were used: FITC-labeled mouse monoclonal anti-␣-SMC actin (clone 1A4, Sigma, St Louis, MO), rat monoclonal anti-mouse Mac-3 (M3/84, Pharmingen, San Diego, CA), HRP-labeled mouse monoclonal antiproliferating cell nuclear antigen (PCNA, PC10, DAKO, Glostrup, Denmark) and rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling technology, Danvers, MA). Detection was performed by a secondary HRP-labeled anti-FITC (DAKO) or by a biotin-labeled anti-rabbit and anti-rat (mouse IgG absorbed, Vector Laboratories, Burlingame, CA) antibody that was developed in combination with the ABC-kit (Vector Laboratories). For demonstration of the complex, 3-amino-9-ethyl-carbazole (AEC) or diaminobenzidine (DAB) was used as a chromogen. Controls without primary antibody were run for each protocol, resulting in consistently negative observations.

The cross-sectional area of the plaques was measured using a computer-assisted image analysis system (Image Pro Plus, Media Cybernetics Inc, Bethesda, MD). For each localization (aortic valves, aortic root, aortic arch, thoracic aorta segment 1 through 5, abdominal aorta; see also Supplementary Fig. 1) plaque area was quantified in one random section. Previous experiments in our laboratory have shown that this was sufficient, because analysis of three sections per localization (at 100 ␮m intervals) did not change statistical power or outcome. Plaque composition (amount of macrophages and SMCs, thickness of the fibrous cap, collagen content), cell replication and amount of apoptosis were measured in plaques at the aortic valves (one random section per mouse). Again, previous experiments in our laboratory demonstrated that this was sufficient. Results were expressed as percent positive area (the total area of the plaque was set as 100%). This blinded analysis was also performed with Image Pro Plus. An intensity threshold mask was defined by sampling and was applied to all specimens. Maximal fibrous cap thickness was measured manually using Image Pro Plus following ␣-SMC actin immunostaining. We only considered fibrous caps that extended to the endothelial cell layer. 2.3. Statistical analysis All results are expressed as mean ± SEM. Statistical analysis was performed by SPSS package for Windows (version 16.0, SPSS Inc., Chicago, IL). The statistical tests are indicated in the legend of the tables. P < 0.05 was considered statistically significant. 3. Results 3.1. Characterization of APP−/− /apoE−/− mice Homozygous APP−/− /apoE−/− mice were fertile and appeared healthy up to 14 months of age. Both the APP−/− and APP−/− /apoE−/− mice showed a reduced body weight as compared to apoE−/− mice (Table 1). Unlike APP−/− mice, apoE−/− and APP−/− /apoE−/− mice fed a Western type diet for 16 weeks showed significantly increased plasma lipid values (Table 1). The levels of total cholesterol were similar in the apoE−/− and APP−/− /apoE−/− mice. Plasma triglycerides were significantly decreased in the APP−/− /apoE−/− mice as compared to the apoE−/− mice (Table 1).

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Fig. 1. Immunohistochemical detection of macrophages (Mac-3), SMCs (␣-SMC actin), collagen (Sirius red), cell replication (PCNA) and apoptotic cells (cleaved caspase-3) in plaques from apoE−/− and APP−/− /apoE−/− mice at the aortic valves after 16 weeks on a Western type diet. AV, aortic valve. Scale bar = 100 ␮m.

3.2. Effects of APP deficiency on atherosclerotic plaque formation and composition ApoE−/− and APP−/− /apoE−/− mice fed a Western-type diet for 16 weeks showed atherosclerotic lesions at the level of the aortic valves, root, arch, distal thoracic aorta (TA segment 5) and abdominal aorta. However, plaque formation was significantly reduced in

the distal thoracic and abdominal aorta of APP−/− /apoE−/− mice (Table 1). In the aortic valves, root and arch, the plaque area was unaffected. Similar results were obtained after 32 weeks of Western-type diet (Supplementary Table 1). APP−/− mice did not develop atherosclerosis. Although the total plaque area at the aortic valves was similar, the area occupied by macrophages (Mac-3 positive) was signifi-

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cantly decreased in APP−/− /apoE−/− mice as compared to apoE−/− mice (Fig. 1, Table 1). There was no significant difference in ␣-SMC actin staining between the two strains (Fig. 1, Table 1). However, we found a significant increase in collagen content (Sirius red positive area; Fig. 1, Table 1) and thickness of the fibrous cap in APP−/− /apoE−/− mice (Table 1). Cell replication was reduced in APP−/− /apoE−/− mice as demonstrated by a significant decrease in PCNA positive area (Fig. 1, Table 1). The number of apoptotic cells (cleaved caspase-3 positive area) was not different between the two strains (Fig. 1, Table 1).

4. Discussion In the present study, we demonstrated that plaque formation in APP−/− /apoE−/− mice was attenuated in the distal thoracic and abdominal aorta as compared to apoE−/− mice. These results indicate that APP deficiency evokes site specific effects on plaque size. It is well-known that APP−/− mice weigh 15–20% less than agematched wild type controls [6]. The body weight deficit can be rescued by soluble APP␣ [6], which exerts proliferative effects [7]. Because the effects of APP-deficiency on plaque area were site specific, changes in body weight or energy homeostasis are unlikely to explain the effect of APP-deficiency on plaque development. It is important to note that the plasma levels of total cholesterol in the apoE−/− mice and the APP−/− /apoE−/− mice were statistically not different. Interestingly, plasma triglycerides were significantly decreased in the APP−/− /apoE−/− mice as compared to the apoE−/− mice. Therefore, the attenuated atherogenesis that we observed in the apoE−/− mice lacking APP may be at least partly explained by the decreased plasma triglyceride levels in these mice. Plaques near the aortic valves were not reduced in size, possibly because they were too advanced. However, these plaques showed a more stable phenotype as demonstrated by a thicker fibrous cap, a decreased number of macrophages and increased collagen content. Because the SMC content was not changed, the latter effect might be related to decreased collagen degradation by macrophages rather than increased production by SMCs. The turnover of macrophages in the plaque is in general determined by the recruitment of monocytes, the rates of cell death and cell proliferation, and emigration. We could demonstrate that the frequency of apoptotic cell death in plaques at the level of the aortic valves was similar in APP−/− /apoE−/− mice and apoE−/− mice. However, cell replication, as assessed by PCNA, was significantly reduced in APP−/− /apoE−/− mice. Because the vast majority of replicating cells in atherosclerotic plaques are macrophages [8], we assume that decreased replication of these cells is an important factor that contributes to the reduced macrophage content in plaques from APP−/− /apoE−/− mice. It is known that monomeric APP can act as a growth factor receptor when bound to the cell membrane or as a growth factor once it is released by ␣-secretase cleavage into the external milieu [9,10]. Furthermore, APP in vascular endothelial cells could have a role in regulating adhesion of monocytes [11], which is one of the earliest detectable stages in atherogenesis [1]. In addition, after integrin-dependent adhesion of monocytes, APP is recruited into a multi-receptor signaling complex that leads to activation of mitogen-activated protein kinases and subsequent production of pro-inflammatory proteins such as COX-2 and IL1␤ [12]. Overall, these data demonstrate that APP promotes plaque inflammation at different levels: it may function as a proinflammatory and growth-stimulating agent and mediates adhesion of monocytes at the endothelium. Besides its role in early atherogenesis, APP is abundantly present in ␣-granules of platelets and is released upon platelet activation [13]. Platelets can enter atherosclerotic plaques by microvessels or intraplaque microhemorrhages [14]. Macrophage activation

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evoked by APP after platelet phagocytosis in advanced atherosclerotic plaques may play a role in plaque destabilization and may ultimately lead to plaque rupture and subsequent atherothrombosis [4–15]. However, necropsy findings in patients with Down’s syndrome, bearing an extra APP copy, have suggested that these individuals are less prone to the development of atherosclerosis. The arteries of patients with Down’s syndrome contain fewer lesions and less calcium than arteries of controls. Even though coronary arteries of Down’s syndrome patients are not completely free of atherosclerosis, it is milder than in patients with other mental diseases or healthy subjects of the same age [16]. In spite of that, other factors than APP may explain the decreased atherogenesis in trisomy 21 patients, because high levels of circulating adiponectin [17] or nerve growth factor [18] might be protective in Down’s syndrome patients. In conclusion, our findings provide evidence that lack of APP attenuates atherogenesis and leads to plaque stability. Currently, there are no drugs available that influence the expression of APP. In contrast, macrophage activation after platelet phagocytosis can be reduced by drugs known to interfere with APP processing enzymes (secretases) or with the progression of Alzheimer’s disease [4], but the cardiovascular effects of these drugs are still largely unknown. APP is cleaved enzymatically by nonamyloidogenic and amyloidogenic pathways. ␣-Secretase cleaves APP within the ␤-amyloid peptide (A␤) sequence, resulting in the release of a secreted fragment of APP and precluding A␤ generation. Cryptotanshinone, an active component of the medicinal herb Salvia miltiorrhiza, improves learning and memory in several models of Alzheimer’s disease. This compound modulates APP metabolism by elevation of ␣-secretase activity [19]. Therefore, an ␣-secretase “agonist” – such as A Disintegrin And Metalloprotease (ADAM)-activator – might also be helpful in the prevention of atherosclerosis. Further insights in the pathophysiology and the pharmacology of APP and its processing in atherosclerosis might contribute to the understanding of present and future treatment strategies in the prevention of atherogenesis and destabilization of atherosclerotic plaques. Acknowledgments This work was supported by the Fund for Scientific Research (FWO)-Flanders (grant no G.0113.06), the University of Antwerp (NOI-BOF) and the Bekales Foundation. The authors are indebted to Rita Van De Bossche, Hermine Fret and Ludo Zonnekeyn for their excellent technical assistance. We dedicate this paper to our secretary, Mrs. Liliane Van den Eynde, who passed away on January 17th, 2011. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2011.01.032. References [1] Lusis AJ. Atherosclerosis. Nature 2000;407(6801):233–41. [2] Van Assche T, Hendrickx J, Crauwels HM, et al. Transcription profiles of aortic smooth muscle cells from atherosclerosis-prone and resistant regions in young apolipoprotein E-deficient mice before plaque development. J Vasc Res 2010;48(1):31–42. [3] Reinhard C, Hébert SS, De Strooper B. The amyloid-beta precursor protein: integrating structure with biological function. EMBO J 2005;24(23): 3996–4006. [4] Jans DM, Martinet W, Van De Parre TJ, et al. Processing of amyloid precursor protein as a biochemical link between atherosclerosis and Alzheimer’s disease. Cardiovasc Hematol Disord Drug Targets 2006;6(1):21–34. [5] Tibolla G, Norata GD, Meda C, et al. Increased atherosclerosis and vascular inflammation in APP transgenic mice with apolipoprotein E deficiency. Atherosclerosis 2010;210(1):78–87.

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