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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Published: May 31st, 2016

DOI:

10.3791/54017

1Anesthesia Center for Critical Care Research of the Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, 2Cardiovascular Research Center and Cardiology Division of the Department of Medicine, Massachusetts General Hospital, 3Cardiovascular Division, Brigham and Women's Hospital, 4Harvard Medical School, 5Department of Anesthesiology, Uniklinik RWTH Aachen, RWTH Aachen University, 6Center for Immunology and Inflammatory Diseases and the Division of Rheumatology, Allergy, and Immunology of the Department of Medicine, Massachusetts General Hospital
* These authors contributed equally

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Cardiovascular disease is the leading cause of morbidity and mortality in the world, including the United States where it accounts for over 780,000 deaths annually.1 Coronary artery calcification and aortic calcification are hallmarks of atherosclerotic disease and serve as strong predictors of cardiovascular events.2-4 Two main types of vascular calcification have been reported in adults: intimal calcification, associated with atherosclerosis, and medial (also known as Mönckeberg) calcification, associated with chronic kidney disease and diabetes.5 Intimal calcification occurs in the setting of lipid accumulation and macrophage i....

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All studies with mice were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Housing and all procedures involving mice described in this study were approved by the Institutional Animal Care and Use Committees of Massachusetts General Hospital (Subcommittee on Research Animal Care). All procedures were performed with care to minimize suffering.

1. Preparation of Reagents

  1. Near-Infrared.......

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Aortic calcification in MGP-/- and wild-type mice was measured using imaging of calcium NIR fluorescence. No calcium NIR signal was detected in the aortas from wild-type mice, indicating the absence of calcification (Figure 2). A strong calcium NIR signal was detected in the aortas from MGP-deficient mice, which is consistent with advanced vascular calcification. Tissue sections of aortas from wild-type and MGP-/- mice were stained with Alizarin red<.......

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Arterial calcification is an important risk factor for cardiovascular disease in humans and may contribute directly to the pathogenesis of cardiovascular events.1,5,52 Intimal calcium deposition in the thin fibrous caps of atherosclerotic disease has been proposed to increase local biomechanical stress and contribute to plaque rupture.53,54 Medial calcification impacts clinical outcomes by increasing arterial stiffness, which can induce cardiac hypertrophy and affect cardiac function.55 T.......

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This work was supported by the Sarnoff Cardiovascular Research Foundation (MFB and TET), the Howard Hughes Medical Institute (TM), the Ladue Memorial Fellowship Award from Harvard Medical School (DKR), the START-Program of the Faculty of Medicine at RWTH Aachen (MD), the German Research Foundation (DE 1685/1-1, MD), the National Eye Institute (R01EY022746, ESB), the Leducq Foundation (Multidisciplinary Program to Elucidate the Role of Bone Morphogenetic Protein Signaling in the Pathogenesis of Pulmonary and Systemic Vascular Diseases, PBY, KDB, and DBB), the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR057374, PBY), the National Institut....

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Name Company Catalog Number Comments
15 ml conical tube Falcon 352096
30 G needle BD 305106
Alpha smooth muscle actin antibody Sigma SAB2500963
Chamber slide Nunc Lab-Tek 154461
Collagenase, Type 2  Worthington LS004176
Dexamethasone Sigma D4902
Dulbecco's Modified Eagle Medium Life Technologies 11965-084
Dulbecco's Phosphate Buffered Saline, no calcium Gibco 14190-144
Elastase Sigma E1250
Fetal bovine serum Gibco 16000-044
Forceps, fine point Roboz RS-4972
Forceps, full curve serrated Roboz RS-5138
Formalin (10%) Electron Microscopy Sciences 15740
Hank's Balanced Salt Solution Gibco 14025-092
Human coronary artery smooth muscle cells PromoCell C-12511
Insulin syringe with needle Terumo SS30M2913
L-ascorbic acid Sigma A-7506
Micro-dissecting spring scissors (13mm) Roboz RS-5676
Micro-dissecting spring scissors (3mm) Roboz RS-5610
NIR, cathepsin (ProSense-750EX) Perkin Elmer NEV10001EX
NIR, osteogenic (OsteoSense-680EX) Perkin Elmer NEV10020EX
Normal Saline Hospira 0409-4888-10
Nuclear fast red Sigma-Aldrich N3020
Odyssey Imaging System Li-Cor Odyssey 3.0
Penicillin/Streptomycin Corning 30-001-CI
Silver nitrate (5%) Ricca Chemical Company 6828-16
Sodium phosphate dibasic heptahydrate Sigma-Aldrich S-9390
Sodium thiosulfate Sigma S-1648
ß-glycerophosphate disodium salt hydrate Sigma G9422
Tissue culture flask, 25 cm2 Falcon 353108
Tissue culture plate (35mm x 10mm) Falcon 353001
Tissue culture plate, six-well Falcon 353046
Trypsin Corning 25-053-CI
Tube rodent holder Kent Scientific RSTR551
Vacuum-driven filtration system Millipore SCGP00525

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