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Medicine

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Published: September 26th, 2018

DOI:

10.3791/58303

1Laboratory of Cardiovascular Endocrinology, IRCCS San Raffaele Pisana, Rome, Italy, 2Department of Human Sciences and Promotion of the Quality of Life, San Raffaele Roma Open University, Rome, Italy

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

Atherosclerosis is one of the main causes of mortality and morbidity worldwide1. It is characterized by a chronic inflammatory state where blood vessels are infiltrated by lipids and leukocytes that determine the formation of atherosclerotic plaques. The majority of acute cardiac events are associated with thrombotic events due to plaque rupture. Plaques prone to rupture are defined "vulnerable" and are characterized by increased infiltration of pro-inflammatory leukocytes, a necrotic core, and a thin fibrous cap2. In the last decades, clinical and experimental studies have clarified the complex pathophysiology o....

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The study was approved by the Italian National Institutes of Health Care and Use Committees, authorization number 493/2016-PR. All procedures were conducted per the guidelines of the European Community for the use of experimental animals (European Directive, 2010/63/UE).

Note: Subcutaneous implantation of osmotic minipump containing vehicle (ethanol in saline solution) or aldosterone (240 µg · kg-1 · d-1) in 8-10 week-old male mice deficient for the Apo.......

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In 8-10 weeks Apo-/- mice, minipumps were implanted to infuse with vehicle or aldosterone (Figure 1E-1I) and fed an atherogenic diet (adjusted calories diet 42% from fat) for 4 weeks. At the end of treatment, mice were euthanized and perfused with PBS and 10% formalin as described above. The aortic root was separated from the apical portion of the heart and was embedded in OCT (Figure 2). Cross section.......

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Atherosclerosis is a chronic inflammatory disorder associated with large and medium vessels involving interactions between multiple cell types, such as macrophages, T-lymphocytes, endothelial cells and smooth muscle cells1. Despite the limitations of murine atherogenic models, a large body of evidence on the atherosclerotic process is available. These models have the advantage of rapidly generating experimental cohorts of a specific age and gender. Mice also show a defined and homogeneous genetic .......

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This work was supported by grants from the Italian Ministry of Health (Ricerca Corrente, GR-2009-1594563 and PE-2011-02347070 to M.C.)

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Name Company Catalog Number Comments
Adjusted calories diet (42 % from fat) Envigo TD.88137 atherogenic diet
Osmotic minipump with filling tube Alzet model 1004 for continous realase
Aldosterone SIGMA A9477 hormone
Ethanol SIGMA 34852-M solvent
Alchol Preps saturated with 70 % Isopropyl Alcohol Kendal Webcol 6818 Disinfectant
Surgery wire (Vicryl 6.0) Demas 500004 surgery
10% Povidone/iodine ointment Aplicare-Meriden 52380-0026-1 Antiseptic
Formalin 10% SIGMA HT5012 to fix vascolature
Cryomold Bio-Optica 07-MP1515 for embedding
O.C.T. Compound Sakura Finetek 4583 for embedding
Cryostat Leica CM1900 instrument for sectioning
Dulbecco's Phosphat Buffered Saline Aurogene AU-L0615-500 buffer solution
Adhesion Slides Polysine VWR 631-0107 microscope glasses
Cover Glasses Bio-Optica 72015 cover glasses
Formaldehyde 37% SIGMA 252549 solvent
Oil Red O solution (0.5 % in isopropanol) SIGMA O1391 staining solution
Mayer’s Hematoxylin SIGMA MHS32 staining solution
Lithium Carbonate SIGMA 62470 washing buffer
Acqueous Mounting Medium Thermo Scientific TA-125-AM mounting solution
Acetone SIGMA 179124 solvent
Phosphomolybdic acid solution SIGMA HT153 for hystology
Direct Red 80 (Picrosirius Red) SIGMA 365548 staining solution
Bio Clear (clearing agent of terpene origin) Bio-Optica 06-1782D product for the preparation of histological samples
Eukitt Quick Hardening mounting medium (Poly(butyl methacrylate-co-methyl methacrylate) SIGMA 3989 mounting solution
Sodium Dodecyl Sulfate Fluka 71725 powder
Hydrogen Peroxide solution (30 %) SIGMA H1009 solution
Vectorstain ABC KIT including: anti-rabbit IgG ABC, normal rabbit serum, Secondary-biotinylated Anti-Rat IgG , Vector Laboratories PK-6100 staining solution
3-amino-9-ethylcarbazole Vector Laboratories SK-4200 staining solution
Mac3 antibody BD Biosciences 553322 antibody
ImagePro Premier 9 Media Cybernetics 050910000-2534 software to analyze images

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