JoVE Journal > Medicine
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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의학

Aort Kalsifikasyon ve Enflamasyon Vasküler düz kas hücrelerinin ve Görüntüleme Kalsifikasyon

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

O yılda 780.000 üzerinde ölüm hesapları nerede kardiyovasküler hastalık Amerika Birleşik Devletleri'nde olmak üzere dünyada morbidite ve mortalitenin önde gelen nedeni vardır. 1 Koroner arter kalsifikasyonu ve aort kalsifikasyon aterosklerotik hastalığın özellikleridir ve kardiyovasküler olayların güçlü belirleyicileri olarak hizmet vermektedir. 2- intimal kalsifikasyon ateroskleroza bağlı ve orta kronik böbrek hastalığı ve diyabet ile bağlantılı kalsifikasyon, (aynı zamanda, Mönckeberg olarak da bilinir) 5 intimal kalsifikasyon lipid birikimi ve makrofaj ortamda oluşur: vasküler kalsifikasyon 4 İki ana tip yetişkin bildirilmiştir. damar duvarı içine sızma. 5,6 Medial duvar kalsifikasyon, bağımsız intimal kalsifikasyon meydana elastin lifleri veya düz kas hücrelerinde lokalize ve lipid birikimi veya makrofaj infiltrasyonu ile ilişkili değildir. 5,7,8 Çalışmaları moleküler mekanizmaları üzerindeVasküler kalsifikasyon hücre bazlı ve hayvan modeli sistemlerinde yararlanmıştır. Medial kalsifikasyon için modeller matriks Gla proteini ile fareler dahil ederken atherocalcific hastalık için kemirgen modelleri, apolipoprotein E (Apo) 9,10 veya düşük yoğunluklu lipoprotein reseptörü (LDLR) 11 yüksek yağlı diyetle beslenen ya eksik fareler dahil (MGP) eksikliği 12 veya yakınındaki total nefrektomi (5/6 nefrektomi modeli) ya da yüksek adenin diyet maruz ya üremi geliştirmek sıçanlar. 13

Burada, MGP eksikliği ile ilişkili vasküler kalsifikasyon modeli odaklanmıştır. MGP arteriyel kalsifikasyon inhibe eden bir hücre dışı bir proteindir. MGP geninde 12 mutasyon Keutel sendromu, brachytelephalangy ek olarak yaygın kıkırdak kalsifikasyon ile karakterize nadir insan hastalığı, işitme kaybı ve periferik pulmoner stenoz tespit edilmiştir. 14-18 olmasa da sık sık gözlenen, 19çoklu arter eş kireçlenme. Keutel sendromu tarif edilmiştir uncarboxylated, biyolojik olarak aktif olmayan mgp yüksek dolaşım düzeyleri kardiyovasküler mortalite tahmin ederken, insan MGP geninde 20 ortak polimorfizmler, koroner arter kalsifikasyon riski, 21-23 ile ilişkilidir. 24. İnsanlarda aksine Keutel sendromlu, MGP eksikliği fareler iki hafta başlayan ve aort rüptürü doğumdan sonra 6-8 hafta die spontan yaygın arteriyel kalsifikasyon oluşan ciddi vasküler fenotip gelişir. 12

ApoE aksine – / – ve LDLR – / – fareler yüksek yağlı diyetle beslenen, ilişkili makrofaj kaynaklı inflamasyon ile intimal vasküler kalsifikasyon geliştirmek, hangi MGP – / -. Fareler makrofajların yokluğunda medial vasküler kalsifikasyon geliştirmek 11,25 rağmen bu bulgular Sex Amaçlı altta yatan farklı uyaranlara önermekal ve medial kalsifikasyon, tümör nekroz faktörü-a ve IL-1 ve ön-osteojenik faktör gibi inflamatuar aracıları içeren vasküler kalsifikasyon katkı tespit edilmiştir kireçlenme. 26 Çoklu sinyal yollarının her iki yapı da aracılık sinyal mekanizmalarında örtüşme vardır Bu çentik Wnt ve kemik morfojenik protein (BMP) sinyal. 27,28 Bunlar sinyal yolları da kemik ilişkili proteinlerin ifadesini artırmak transkripsiyon faktörlerinin solucan ile ilgili transkripsiyon faktörü 2 (Runx2) ve Osterix ekspresyonunu (geliştirmek . / – – ve LDLR – / – fareleri, yüksek yağlı diyet ve kendiliğinden beslenen örneğin kalsifikasyon aracılık damar sisteminde, kemik sklerostin ve alkalin fosfataz) 28-30 Biz ve diğerleri ApoE gözlemlenen vasküler kalsifikasyon göstermiştir / – – MGP gözlenen vasküler kalsifikasyon tüm kemik morfogenetik protein bağlıdır farelerin (BMP) signaling ve burada odaklanmıştır, bu yoldur. 11,25,31 BMP kemik oluşumu için gerekli olan güçlü bir osteojenik faktör, insan ateroskleroz içinde arttırılmış ekspresyon sergileyen bilinmektedir. 32-34 in vitro çalışmalar, düzenlenmesinde BMP sinyal rol var bu Runx2 olarak osteojenik faktör sentezlenmesi. BMP ligandının 35-37 aşırı ekspresyonu, BMP-2, yüksek yağlı diyet ile beslenen ApoE eksikliği olan farelerde vasküler kalsifikasyon gelişimini hızlandırır. 38 Ayrıca, bu tür inhibitörleri sinyal belirli bir BMP kullanımını / – – 39,40 ve / veya LDN-193189 (LDN) olarak ALK3-Fc hem LDLR vasküler kalsifikasyon gelişimini engeller fareler yüksek yağlı diyet ve MGP eksikliği fareler beslenen 11,25.

Vasküler düz kas hücreleri (VSMC'Ier) vasküler kalsifikasyon gelişiminde önemli bir role sahiptir. 30,41,42 gelişen vasküler kalsifikasyon MGP yoksun fareler karakteristik olanbir osteojenik fenotipi VSMC'Ierin bir transdiferansiyonun tarafından terized. Böyle Runx2 ve osteopontin olarak osteojenik belirteçleri eşlik eden bir artış ile myocardin ve alfa düz kas aktin içeren VSMC belirteçlerin azalmış ifadesi MGP sonuçlarının kaybı. Bu değişiklikler vasküler kalsifikasyon gelişimi ile örtüşmektedir. 25,43,44

Aort kalsifikasyon ve farelerde iltihap tipik olarak geç kireçlenme erken kireçlenme ve osteojenik aktivite, von Kossa ve Alizarin kırmızı boyama için alkalin fosfataz aktivitesi ve makrofaj protein belirteçlerinin (örn. Hedef immünohistokimyasal protokoller olarak histokimyasal teknikler kullanılarak değerlendirilir, CD68, F4 / 80, Mac-1, Mac-2, Mac-3). 9,45 Bununla birlikte, bu, standart görüntüleme teknikleri, örnekleme yanlılığa alıcı ve kusurlu zaman enine bölüme aort dokularının işlenmesini gerektirir ve sınırlıdır kendi inflamasyon ve calcificat sayısal yeteneğiBütün aort iyon. Bu protokol, aynı zamanda sağlanan. Görselleştirmek ve bütün aort ve orta ölçekli arteryel kalsiyum ve yakın kızılötesi floresan (NIR) moleküler görüntüleme ex vivo olarak kullanılarak makrofaj birikimini ölçmek için bir yöntem tarif hasat ve farelerden primer aort VSMC'Ierin kültürlenmesi ve uyarılması için bir yöntemdir için sıçangil ve in vitro olarak insan VSMC'Ierinin kalsifikasyon vasküler kalsifikasyon altında yatan moleküler mekanizmaların belirlenmesi. Bu teknikler, in vivo ve atherocalcific hastalığı eğitimi için in vitro yöntemleri hem araştırmacı sağlar.

Protocol

fare ile tüm çalışmalar Ulusal Sağlık Enstitüleri Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu önerilerine sıkı göre yapıldı. Konut ve bu çalışmada açıklanan fareler ile ilgili tüm işlemler (Araştırma Hayvan Bakım Alt Komitesi) Kurumsal Hayvan Bakım ve Kullanım Komiteleri Massachusetts General Hastanesi tarafından kabul edildi. Tüm prosedürler acıyı azaltmak için özenle yapıldı. Reaktiflerin 1. Hazırlık Tüm aortalarının Near-Infrared…

Representative Results

– / – Aort MGP kireçlenme ve vahşi tip fare, kalsiyum NIR floresans görüntüleme kullanılarak ölçülmüştür. Resim kalsiyum NIR sinyal kalsifikasyon yokluğu (Şekil 2) gösteren, vahşi tip farelerden aorta saptandı. Güçlü bir kalsiyum NIR sinyali ileri vasküler kalsifikasyon ile tutarlıdır MGP eksikliği olan farelerde, gelen aorta saptandı. Yabani tip ve gelen aorta doku kesitleri MGP – / – vahşi tip farelerde tespit Resim kalsi…

Discussion

Arter kalsifikasyon insanlarda kardiyovasküler hastalık için önemli bir risk faktörüdür ve kardiyovasküler olayların patogenezinde doğrudan katkıda bulunabilir. Aterosklerotik hastalığın ince fibröz kapaklar 1,5,52 intimal kalsiyum birikimi yerel biyomekanik stresi arttıran ve katkıda bulunmak için öne sürülmüştür plak rüptürü. kardiyak hipertrofi neden ve kalp fonksiyonunu etkileyebilir arteriyel sertlik, artırarak 53,54 medial kalsifikasyon etkileri klinik sonuçları….

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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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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