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Médecine

血管平滑筋細胞と大動脈石灰化と炎症のイメージングの石灰化

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

心血管疾患は、それが毎年78万を超える死者を占める米国をはじめ、世界における罹患率および死亡率の主要な原因である。1冠動脈石灰化と大動脈石灰化は、アテローム性動脈硬化症の特徴であると心血管イベントのような強力な予測因子を提供します。2- 、内膜石灰化アテローム性動脈硬化症と関連しており、内側には、慢性腎臓病と糖尿病に関連する石灰化、（もMönckebergとして知られている）5内膜石灰化は、脂質蓄積とマクロファージの設定で発生します。血管石灰化の4つの主なタイプは、成人で報告されています。血管壁への浸潤。5,6内側側の壁の石灰化は、エラスチン繊維または平滑筋細胞に局在し、脂質沈着やマクロファージの浸潤に関連付けられていない、独立して内膜石灰化の発生。の分子機構に関する研究5,7,8血管石灰化は、細胞ベースおよび動物モデル系に依存していました。 atherocalcific疾患のための齧歯類モデルは、アポリポタンパク質E（アポE）9,10または低密度リポタンパク質受容体（LDLR）中膜石灰化のためのモデルは、行列のGlaタンパク質（MGP）欠損マウスを含むながら11は 、高脂肪食を与えいずれかを欠損したマウスを含みます12またはそれに近い総腎摘出（5/6日の腎摘出モデル）によって、または高アデニン食事療法への曝露のいずれかによって尿毒症を発症ラット。13

ここでは、MGPの欠損に関連する内側血管石灰化のモデルは、に焦点を当てています。 MGPは、動脈石灰化を阻害し、細胞外タンパク質である。MGP遺伝子における12の変異がKeutel症候群、brachytelephalangyに加えて、拡散軟骨石灰化によって特徴づけられるまれなヒト疾患、難聴、および末梢肺動脈狭窄症で同定されている。14-18ではないが、しばしば観察、19複数の動脈の同心円状石灰化がKeutel症候群に記載されている。非カルボキシル化、生物学的に不活性なMGPの高い循環レベルは、心血管死亡率を予測しながら、人間のMGP遺伝子における20の一般的な多型は冠動脈石灰化、21-23リスクの増加と関連している。24人間とは異なり、 Keutel症候群で、MGP欠損マウスは生後2週間で開始自発的な広範囲の動脈石灰化からなる重度の血管の表現型を開発し、原因大動脈破裂に6-8週間出産後に死亡する。12

アポEとは異なり– / –およびLDLR – / –マウスは、高脂肪食を与え、関連マクロファージ誘発性炎症と内膜血管石灰化を開発する、MGP – / – 。マウスは、マクロファージの浸潤が存在しない状態で内側血管石灰化を開発11,25が、これらの知見は、intimに異なる根本的な刺激を示唆していますアルと中膜石灰化は、例えば、腫瘍壊死因子αおよびIL-1およびプロ骨形成因子などの炎症性メディエーターを含む血管石灰化に貢献同定されている石灰化。26、複数のシグナル伝達経路の両方の形式を仲介するシグナル伝達機構で重複がありますこのようなノッチは、Wnt、および骨形成タンパク質（BMP）シグナル。27,28のように、これらのシグナル伝達経路は、次に、骨関連タンパク質の発現を増加させる転写因子ラント関連転写因子2（Runx2の）及びオステリックスの発現を（増加させます– / –およびLDLR – / –マウスは、高脂肪食および自発を与え、例えば石灰化を媒介する血管系において、オステオカルシン、スクレロスチン、およびアルカリホスファターゼ）28-30我々は、他のアポEで観察された血管石灰化のことを実証しましたMGPに観察血管石灰化– / –マウスはすべて、骨形成タンパク質（BMP）SIに依存しますgnaling、それはここに焦点を当てているこの経路である。11,25,31 BMPは、骨形成のために必要な強力な骨形成因子であり、ヒトのアテローム性動脈硬化症で発現増加を示すことが知られている。32-34 in vitro試験を調節におけるBMPシグナル伝達を関与していますなどのRunx2などの骨形成因子の発現。BMPリガンドの35-37過剰発現は、BMP-2は、高脂肪食給餌したApoE欠損マウスにおける血管石灰化の開発を加速。38また、このようなシグナル伝達阻害剤、特定のBMPの使用を39,40 LDN-193189（LDN）として、および/ ​​またはALK3-Fcは、両方のLDLR内の血管石灰化の発症を予防する– / –マウスは、高脂肪食とMGP欠損マウスを与え11,25。

血管平滑筋細胞（VSMC）は、血管石灰化の発展に重要な役割を持っている。MGP欠損マウスに発症30,41,42内側血管石灰化はCHARACです骨形成表現型へのVSMCの分化転換によってterized。このようなRunx2のおよびオステオポンチンなどの骨形成マーカーの同時上昇とミオカルディンおよびα平​​滑筋アクチンを含むVSMCマーカーの発現低下でMGP結果の損失、。これらの変化は、血管石灰化の発達と一致する。25,43,44

大動脈石灰化と炎症マウスでは、典型的には、早期の石灰化および骨形成活性のためのアルカリホスファターゼ活性などの組織化学的技術を利用して評価され、後半に石灰化のためのフォン・コッサおよびアリザリンレッド染色、およびマクロファージのタンパク質マーカー（ 例えば 、標的免疫組織化学プロトコル。、CD68、F4 / 80、のMac-1、のMac-2 MAC-3）9,45しかし、これらの標準的な画像化技術が原因で、サンプリングの偏りに時間がかかり、不完全である、断面に大動脈組織の処理を必要とし、それらに限定されています炎症とcalcificatを定量する能力全体の大動脈内のイオン。このプロトコルは、ex vivo近赤外蛍光（NIR）分子イメージングを利用した全大動脈および中型動脈石灰化およびマクロファージの蓄積を可視化し、定量化する方法を説明している。また、提供収穫するための方法であるとマウスからの一次大動脈のVSMCを培養し、誘導します血管石灰化の根底にある分子メカニズムを決定するために、マウスおよびインビトロでヒトのVSMCの石灰化。これらの技術は、インビボおよびatherocalcific疾患を研究するためのインビトロ方法で両方で調査員を提供しています。

Protocol

マウスを用いた研究はすべて国立衛生研究所の実験動物の管理と使用に関するガイドの推奨事項に厳密に従って実施しました。住宅この研究で説明したマウスに関連するすべての手続きは、マサチューセッツ総合病院（研究動物管理部会）の施設内動物管理使用委員会によって承認されました。すべての手順は、苦痛を最小限にするために注意して行きました。 試薬の調?…

Representative Results

– / -大動脈MGPにおける石灰化および野生型マウスは、カルシウムNIR蛍光イメージングを用いて測定しました。いいえカルシウムNIR信号は、石灰化が存在しない（ 図2）を示す、野生型マウス由来の大動脈において検出されませんでした。強力なカルシウムNIR信号が高度な血管石灰化と一致しているMGP欠損マウスからの大動脈で検出されました。野?…

Discussion

動脈石灰化は、ヒトにおける心血管疾患の重要な危険因子であり、心血管イベントの発症に直接貢献するかもしれない。アテローム性動脈硬化症の薄い線維性キャップで1,5,52内膜カルシウム沈着は、ローカル生体力学的ストレスを増加させ、に貢献することが提案されていますプラーク破裂。心臓肥大を誘導し、心臓機能に影響を与える可能性動脈硬化を増加させることによって…

Divulgations

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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Tags

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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