We-W37:2 Unstable plaque in laboratory animals: The apolipoprotein E knockout mouse fed a high-fat diet

We-W37:2 Unstable plaque in laboratory animals: The apolipoprotein E knockout mouse fed a high-fat diet

Wednesday, June 21, 2006: Workshop We-W37 Anintal models (lstpart) I W e - W 3 7 : 2 I U N S T A B L E P L A Q U E IN L A B O R A T O R Y A N I M A L ...

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Wednesday, June 21, 2006: Workshop We-W37 Anintal models (lstpart) I W e - W 3 7 : 2 I U N S T A B L E P L A Q U E IN L A B O R A T O R Y A N I M A L S : T H E APOLIPOPROTEIN E KNOCKOUT MOUSE FED A HIGH-FAT D I E T J.L. Johnson. Bristol Heart lnstitute, Universi~ of Bristol, Bristol, England

Objective: Atherosclerotic plaque rupture, with subsequent occlusive thrombosis, is the underlying cause of most cases of sudden cardiac death. Scientists have therefore attempted to develop animal models that mimic atherosclerotic plaque rupture, to allow direct interrogation of the mechanisms involved in this complex pathology. The apolipoprotein E knockout mouse is a well established animal model of hypercholesteroleamia. After 6 months on a high-fat-diet, advanced fibrous plaques axe observed with thin lumenal elastin and collagen fibres capping small lipid-rich globular lesions, suggesting that the plaques may be vulnerable to rupture. Due to the apparent fragility and vulnerability of these lesions, we decided to high-fat feed apolipoprotein E knockout mice for up to 12 months, and survey the major arteries and their branch points for evidence of plaque rupture. Methods: Apolipoprotein E knockout mice were fed a high-fat, cholesterolenriched diet from the age of 8 weeks. Animals were terminated at intervals between 4 weeks and 36 weeks of fat-feeding. This animal model was further characterised by assessing the effects of statin therapy (pravastatin) on atherosclerotic plaque development and stability. Results: Apolipoprotein E knockout mice were found to develop large complex atherosclerotic lesions throughout the aorta after prolonged highfat feeding. Interestingly, atherosclerotic plaques within the brachiocephalic artery showed signs of acute plaque rupture [1]. Further investigation revealed that even short periods of high-fat feeding (eight weeks) resulted in complex plaques with healed plaque ruptures that could be utilised as a marker of plaque instability and rupture [2]. The healed plaque ruptures were characterised by the presence of structures rich in elastin, smooth muscle cells and contained areas of fibrin deposition, and were classified as buried fibrous caps [3]. Treatment with pravastatin reduced the number of the buried fibrous caps [3]. Because inhibition of plaque progression by pravastatin could indirectly reduce the number of plaque ruptures that occur, we also commenced treatment in animals that had already been fat-fed for 16 weeks. This reduced the incidence of acute plaque rupture and the incidence of sudden death. These data suggest that the effects observed mirror those reported in humans, further illustrating that the fat-fed apoE knockout mouse is a useful model of plaque rupture. Conclusions: Plaque rupture occurs at high frequency in the brachiocephalic arteries of male apolipoprotein E knockout mice after 8 weeks of fat-feeding, pravastatin treatment inhibits early plaque rupture, and is also effective when commenced after unstable plaques have already developed. these data suggest that the fat-fed apolipoprotein E knockout mouse is a reproducible and reliable model of plaque rupture. References [1] Johnson JL, Jackson CL. Atherosclerotic plaque rupture in the apolipoprotein E knockout mouse. Atherosclerosis 2001;154:399-406. [2] Williams H, Johnson JL, Carson KGS, Jackson CL. Characteristics of intact and ruptured atherosclerotic plaques in brachiocephalic arteries of apolipoprotein E knockout mice. ArteHoscler Thromb Vasc Biol. 2002;22:788-792. [3] Johnson J, Carson K, Williams H, Karanam S, Newby A, Angelini G, George S, Jackson C. Plaque rupture after short periods of fat-feeding in the apolipoprotein E knockout mouse: model chaxacterisation, and effects of pravastatin treatment. Cireulation 2005; 111:1422-1430. Funding: British Heart Foundation

I We-W37:3 I AUTOSOMAL R E C E S S I V E H Y P E R C H O L E S T E R O L E M I A (ARH) KNOCKOUT M O U S E HAS DELAYED CATABOLISM OF LDL IN V I V O BUT N O R M A L I N T E R N A L I Z A T I O N OF LDL IN VITRO M. Harada-Shiba I , A. Takagi I , K. Marutsuka 2 , S. Moriguchi 2, H. Yagyu 3, S. Ishibashi 3 , Y. Asada 2, S. Yokoyama 4 . 1National Cardiovascular Center

Researeh Institute, Suita, Osaka, Japan." 2Miyazaki Medical College, Mivazaki,~ Mivazaki,~ Japan: ~Jtcht" "Medtcal" College, Kawachi-Gun, Tochigi, Japan." 4Nagoya Ci~ Universi~, Nagoya, Aichi, Japan Objective: We previously chaxacterized the patients with A R H as having severe hypercholesterolemia and retarded plasma LDL cleaxance despite the normal LDL receptor function in their cultured fibroblasts, and identified a mutation in A R H locus in these patients. A R H protein is an adaptor protein of the LDL receptor, and reportedly modulates its internalization. In order


to study the function of this protein, we developed A R H knockout mice (ARH-/-). Metbods and Results: Plasma total cholesterol level was higher in ARH-/mice than that in wild-type mice (ARH+/+) being attributed to a 6-fold increase of LDL. Clearance of 125I-LDL from plasma was retarded in ARH/-mice, as mush as that found in LDLR-/- mice. Fluorescence activity of the intravenously injected DiI-LDL was recovered in the cytosol of the hepatocytes of ARH+/+ mice, but not in those of ARH-/- or LDLR-/- mice. Also, less amount of radioactivity was recovered in the liver of ARH-/- or LDLR-/mice when [3H]cholesteryl oleoyl ether (CE)-labeled LDL was injected. In contrast, uptakes of [3H]CE-labeled LDL, 125I-LDL and DiI-LDL were all normal or slightly subnormal when the ARH-/- hepatocytes were cultured. Conclusion: The function of the hepatic LDL receptor is impaired in the ARH-/- mice in vivo despite its normal function in vitro. These findings were consistent with the observations with the A R H homozygous patients, and suggested that certain cellular environmental factors modulate the requirement of A R H for the LDL receptor function.

W e - W 3 7 : 4 ] I N C R E A S E D A C T I V I T Y OF ABCA1 P R O T E C T S

AGAINST ATHEROSCLEROSIS R.R. Singaraia 1, C. Fievet 2, M.R. Hayden 1 . 1 Universi~ of British Columbia,

Vancouver; Canada: 21nstitut Pasteur de Lille, Lille, Cedex, France Significant controversy exists as to the role of ABCA1 in atherosclerosis. Others have shown that expression of ABCA1 on the LDLr-/- background results in a pro-atherogenic lipid profile and increased atherosclerosis and have also shown increased atherosclerosis when A B C A l w a s expressed in ApoE-/mice. We previously showed significant protection agadnst atherosclerosis when ABCA1 was expressed in ApoE-/- mice. To address A B C A l s role in atherosclerosis, we expressed human ABCA1 in LDLr-/- mice and in mice lacking both LDLr and endogenous mouse ABCA1 (LDLr-/-mABCA1-/-). Expression of ABCA1 in LDLr-/- mice resulted in significantly decreased aortic lesions on a western type diet (wtd) (LDLr-/-: 491.0-4-173.3 vs ABCAI+LDLr-/-: 211.5-4-115.4 m m 2, n=5, p=0.02). No differences in lesions were observed on the chow diet. In addition, no significant differences in plasma lipid levels were observed on both chow and wtd indicating that ABCA1 is able to protect against atherosclerosis independent of plasma HDL-C levels. Expression of ABCA1 in LDLr-/-mABCA1-/- double knockout mice resulted in a significant reduction in atherosclerotic lesions on both chow (LDLr-/-mABCA1-/-: 18.2-4-6.5 vs. ABCAI+LDLr-/-mBCA1-/-: 4.0-4-1.0 m m 2, n=4, p=0.02) and wtd (LDLr-/-mABCA1-/-: 1560.2-4-143.5 vs. ABCAI+LDLr-/-mBCA1-/-: 516.2-4-313.4mm-, n=4, p=0.02). In addition, expression of ABCA1 in the LDLr-/-mABCA1-/- background resulted in significant increase in plasma total cholesterol and HDL-C both on chow and wtd. Together, these data provide further support that expression of ABCA1 results in significant protection against atherosclerosis in different genetic models.

We-W37:5 ] MACROPHAGE ABCB4 D E F I E N C Y PROMOTES ATHEROSLCEROTIC L E S I O N D E V E L O P M E N T IN LDLR D E F I C I E N T M I C E M. Pennings 1 , R.B. Hildebrand 1 , T.J.C. Van Berkel 1 , A.K. Groen-, M. Van Eck 1. 1Div. of Biopharntaceutics, Leiden, the Netherlands." "-Dept. of

Gastrointestinal and Liver Diseases, Amsterdam, the NetherlatMs Objectives: In addition to A B C A 1, a well-known cholesterol and phospholipid transporter, macrophages express several other ABC-transporters, including ABCB4. ABCB4 promotes the transport of phospholipids. The aim of this study was to determine the role of macrophage ABCB4 in atherosclerotic lesion development. Methods: LDL receptor knockout mice were transplanted with bone marrow from ABCB4 knockout (KO) or wild-type mice to generate chimeras that specifically lack ABCB4 in macrophages. Atherosclerotic lesion development was induced by feeding Western-type diet (15% fat and 0.25% cholesterol) for 9 weeks. Results: On Western-type diet serum cholesterol levels were 22% lower in mice reconstituted with ABCB4 KO bone marrow (1642-4-98 mg/dl for ABCB4 KO and 2112-4-484 mg/dl for wild-type transplanted mice, p=0.049), due to lower VLDL and LDL cholesterol levels. Despite the lower serum cholesterol levels, ABCB4 deficiency on macrophages resulted in a two-fold (p=0.0053) increase in lesion size (5.79-4-0.2 • 103 in ABCB4 KO transplanted mice compared to 3.17-4-1.6• 103 i~m2 in mice reconstituted with wild-type bone marrow). In vitro foam cell formation induced with acetylated LDL was

XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006