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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 Fluorescence Imaging of Whole Aortas
   Note: A bisphosphonate-derived, near-infrared fluorescent imaging probe can be used to mark osteogenic ac....

Results

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<.......

Discussion

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.......

Materials

List of materials used in this article

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 (13 mm)	Roboz	RS-5676
Micro-dissecting spring scissors (3 mm)	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 (35 mm x 10 mm)	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