Published: May 31st, 2016
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....
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
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<.......
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.......
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....
|15 ml conical tube
|30 G needle
|Alpha smooth muscle actin antibody
|Collagenase, Type 2
|Dulbecco's Modified Eagle Medium
|Dulbecco's Phosphate Buffered Saline, no calcium
|Fetal bovine serum
|Forceps, fine point
|Forceps, full curve serrated
|Electron Microscopy Sciences
|Hank's Balanced Salt Solution
|Human coronary artery smooth muscle cells
|Insulin syringe with needle
|Micro-dissecting spring scissors (13mm)
|Micro-dissecting spring scissors (3mm)
|NIR, cathepsin (ProSense-750EX)
|NIR, osteogenic (OsteoSense-680EX)
|Nuclear fast red
|Odyssey Imaging System
|Silver nitrate (5%)
|Ricca Chemical Company
|Sodium phosphate dibasic heptahydrate
|ß-glycerophosphate disodium salt hydrate
|Tissue culture flask, 25 cm2
|Tissue culture plate (35mm x 10mm)
|Tissue culture plate, six-well
|Tube rodent holder
|Vacuum-driven filtration system
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