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Abstract
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Protocol
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医学

主动脉钙化和炎症血管平滑肌细胞和成像的钙化

Published: May 31, 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

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

心血管疾病是发病率和死亡率在世界上的首要原因，包括美国在那里它占每年超过78万例死亡。1冠状动脉钙化和主动脉钙化是动脉粥样硬化性疾病的标志，服务心血管事件的强预测因子。2- 4两种主要类型的血管钙化已经报道在成人：内膜钙化，与动脉粥样硬化有关，和内侧（也称为Mönckeberg）钙化，慢性肾脏疾病和糖尿病相关的脂质积累和巨噬细胞的设置发生5内膜钙化。浸润到血管壁。5,6-内侧壁钙化发生独立的内膜钙化，定位于弹性蛋白纤维或平滑肌细胞，并且不与脂质沉积或巨噬细胞浸润有关。5,7,8研究的分子机制血管钙化都依赖细胞系和动物模型系统。对于atherocalcific疾病的啮齿动物模型包括小鼠在任一载脂蛋白E（ApoE基因）9,10或低密度脂蛋白受体（LDLR）11喂食高脂肪食物不足，而型号为膜钙化包括与基质Gla的蛋白质的小鼠（MGP）缺乏症12，或任一由邻近总肾切除术（5/6肾切除模型）或通过暴露于高腺嘌呤饮食开发尿毒症大鼠13

在这里，MGP缺乏相关的内侧血管钙化模型的重点是。 MGP是抑制动脉钙化的细胞外蛋白。在MGP基因12的突变已在Keutel综合征，一种罕见的人类疾病除了brachytelephalangy特征在于漫射软骨钙化，听力丧失，外围肺动脉狭窄已确定。14-18虽然未经常观察，19多动脉钙化同心已经Keutel综合征24被描述。同时，未羧化，无生物活性MGP更高的循环水平预测心血管疾病的死亡率在人类基因MGP 20种常见多态性与冠状动脉钙化风险增加，21-23有关。与人类不同与Keutel综合征，MGP缺陷小鼠制定严重的血管型自发组成的广泛动脉钙化的开始两周年龄和出生后死亡6-8周因主动脉破裂。12

不像的ApoE – / –和LDLR – / –小鼠饲喂高脂肪的饮食，其发展与相关巨噬细胞诱导的炎症内膜血管钙化，MGP – / –小鼠发展内侧血管钙化在没有巨噬细胞浸润的11,25虽然这些研究结果表明为intim不同的潜在刺激人及内侧钙化，有在介导有助于血管钙化包括炎症介质例如肿瘤坏死因子α和IL-1和促成骨因子钙化。26多个信号通路已经鉴定的两种形式的信号机制重叠如缺口，Wnt信号，和骨形态发生蛋白（BMP）信令。27,28，这些信号传导途径增加了转录因子侏儒相关转录因子2（Runx2的）和osterix的，这反过来又增加骨相关蛋白表达的表达（ 如 ：，骨钙素，硬化，和碱性磷酸酶）在介导钙化脉管28-30我们和其他人已经表明，在ApoE基因中观察到的血管钙化– / –和LDLR – / –小鼠喂食高脂肪饮食和自发/ – –在MGP观察血管钙化小鼠都依赖于骨形态发生蛋白（BMP）SIgnaling，它是这一途径，重点是在这里。11,25,31 BMP是骨形成所需的强效的成骨因子和已知呈现在人类动脉粥样硬化的增加的表达。32-34 体外研究在调节牵连BMP信号成骨因子如Runx2的表达。35-37表达的BMP配体，BMP-2，加速血管钙化中的ApoE缺陷小鼠发展喂食高脂肪的饮食。38此外，信令抑制剂例如使用特定的BMP作为LDN-193189（LDN）39,40和/或ALK3-Fc的防止血管钙化的发展都LDLR – / –小鼠喂食高脂肪的饮食和MGP缺陷小鼠11,25。

血管平滑肌细胞（VSMC）在血管钙化的发展具有关键作用。30,41,42内侧血管钙化，在开发MGP缺陷小鼠是CHARAC由血管平滑肌细胞的转terized到成骨表型。 MGP的结果，包括心肌素和α-平滑肌肌动蛋白VSMC标志物的表达减少损失，具有成骨标记，如Runx2的骨桥蛋白和随之而来的上升。这些变化与血管钙化的发展相吻合。25,43,44

主动脉钙化和小鼠的炎症通常是评估利用组织化学技术，如早期钙化和成骨活性，冯·科萨和茜素红染色后期钙化碱性磷酸酶活性，并针对巨噬细胞蛋白标志物（ 如免疫组化协议，CD68，F4 / 80，苹果-1，Mac的-2，Mac的-3）。9,45然而，这些标准成像技术需要主动脉组织成横截面的处理，这是耗时和不完善由于抽样偏差，并且被限制了它们的量化炎症和calcificat能力离子在整个主动脉。这个协议描述的方法来可视化和量化整个主动脉和中型动脉钙化和利用近红外荧光（NIR）分子成像体外的巨噬细胞积聚。还提供了用于收集和小鼠培养初级主动脉平滑肌细胞和诱导的方法为了鼠和体外人平滑肌钙化来确定血管钙化的分子机制。这些技术提供了研究者在体内和在研究atherocalcific疾病的体外方法用。

Protocol

严格按照指南中的建议为美国国立卫生研究院的实验动物的护理和使用进行小鼠所有的研究。住房和涉及本研究中所描述的小鼠的所有程序是由马萨诸塞州总医院的机构动物护理和使用委员会（小组委员会研究动物保健）的批准。所有的程序都小心进行，以尽量减少痛苦。 1.试剂的制备所有主动脉的近红外荧光成像注：A二膦酸盐衍生的，近红外荧光成像探针可用…

Representative Results

/ – -在MGP主动脉钙化和野生型小鼠中使用钙NIR荧光成像测定。在从野生型小鼠的主动脉中没有检测到钙的NIR信号，指示不存在钙化的（ 图2）。从MGP缺陷小鼠，这是与先进的血管钙化一致主动脉检测到强烈的钙NIR信号。从野生型和主动脉的组织切片MGP – / -小鼠用茜素红25（ 图3A – B）中染色，证实在MGP缺陷小鼠?…

Discussion

动脉钙化是人类的心血管疾病的重要危险因素，并可能直接向心血管事件的发病贡献。在动脉粥样硬化性疾病的薄纤维帽1,5,52内膜钙沉积已提出增加本地生物力学应力并有助于斑块破裂。通过增加动脉僵硬度，从而可诱发心肌肥厚，影响心脏功能53,54膜钙化影响临床疗效。55因此，了解背后血管钙化将提供重要的见解人类疾病和可能确定新的目标的分子机制治疗。

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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 Institute of Diabetes and Digestive and Kidney Diseases (R01DK082971, KDB and DBB), the American Heart Association Fellow-to-Faculty Award #11FTF7290032 (RM), and the National Heart, Lung, and Blood Institute (R01HL114805 and R01HL109506, EA; K08HL111210, RM).

Materials

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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Keywords: Vascular CalcificationAortic CalcificationVascular Smooth Muscle CellsAortaIn VivoEx VivoInflammationMacrophageAtherosclerosisCalciumCathepsinImagingFluorescence Imaging
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O’Rourke, C., Shelton, G., Hutcheson, J. D., Burke, M. F., Martyn, T., Thayer, T. E., Shakartzi, H. R., Buswell, M. D., Tainsh, R. E., Yu, B., Bagchi, A., Rhee, D. K., Wu, C., Derwall, M., Buys, E. S., Yu, P. B., Bloch, K. D., Aikawa, E., Bloch, D. B., Malhotra, R. Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation. J. Vis. Exp. (111), e54017, doi:10.3791/54017 (2016).
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