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Introduction
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医学

Verkalking van vasculaire gladde spiercellen en Imaging van Aorta verkalking en Ontsteking

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

Hart- en vaatziekten zijn de belangrijkste oorzaak van morbiditeit en mortaliteit in de wereld, waaronder de Verenigde Staten, waar het goed is voor meer dan 780.000 doden per jaar. 1 Coronaire verkalking en aorta verkalking zijn kenmerken van atherosclerose en dienen als sterke voorspellers van cardiovasculaire gebeurtenissen. 2- 4 Twee belangrijke types van vasculaire calcificatie zijn gemeld bij volwassenen: intima verkalking, geassocieerd met atherosclerose, en mediale (ook bekend als Monckeberg) verkalking, geassocieerd met chronische nierziekte en diabetes 5 intima verkalking optreedt in de setting van lipide-accumulatie en macrofagen. infiltratie in de vaatwand. 5,6 mediale wand verkalking optreedt onafhankelijk van intimale verkalking, lokaliseert elastine vezels of gladde spiercellen en is niet geassocieerd met lipide afzetting of macrofaag infiltratie. 5,7,8 Studies over de moleculaire mechanismen vanvasculaire calcificatie hebben vertrouwd op basis van cellen en dierlijke modelsystemen. Knaagdier modellen voor atherocalcific ziekte zijn deficiënte muizen in beide apolipoproteïne E (ApoE) 9,10 of low-density lipoprotein receptor (LDLR) 11 gevoed een vetrijk dieet, terwijl de modellen voor de mediale verkalking onder andere muizen met matrix Gla proteïne (MGP) deficiëntie 12 of ratten die uremie ontwikkelen op bijna totale nefrectomie (de 5 / 6e nefrectomie model) of door blootstelling aan een hoge-adenine dieet. 13

Hier wordt het model van mediale vasculaire calcificatie geassocieerd met MGP deficiëntie gericht op. MGP is een extracellulair eiwit dat aderverkalking remt. 12 Mutaties in het MGP gen geïdentificeerd in Keutel syndroom, een zeldzame menselijke ziekte gekenmerkt door diffuse kraakbeen verkalking naast brachytelephalangy, gehoorverlies, en perifere pulmonale stenose. 14-18 Hoewel niet vaak waargenomen, 19concentrische verkalking van meerdere slagaders is beschreven in Keutel syndroom. 20 Common polymorfismen in de humane MGP-gen worden geassocieerd met een verhoogd risico op coronaire calcificaties, 21-23 terwijl hogere niveaus van circulerende uncarboxylated, biologisch inactief MGP cardiovasculaire mortaliteit voorspellen. 24 In tegenstelling tot mensen met Keutel syndroom, MGP-deficiënte muizen ontwikkelen ernstige vasculaire fenotype bestonden uit spontane wijdverbreide aderverkalking vanaf twee weken oud en sterven 6-8 weken na de geboorte door aortaruptuur. 12

Unlike ApoE – / – en LDLR – / – muizen gevoed een vetrijk dieet, dat intimale vasculaire calcificatie met geassocieerde macrofaag geïnduceerde inflammatie ontwikkelen, MGP – / -. Muizen ontwikkelen mediale vasculaire calcificatie in afwezigheid van macrofaag infiltratie 11,25 Hoewel deze bevindingen suggereren verschillende onderliggende stimuli voor intimal en mediale verkalking, er overlap in de signalering mechanismen die beide vormen van calcificatie. 26 meerdere signaleringsroutes geïdentificeerd die bijdragen tot vasculaire calcificatie inclusief inflammatoire mediatoren zoals tumor necrosis factor-α en IL-1 en pro-osteogene factoren bemiddelen zoals Notch, Wnt, en bot morfogenetisch eiwit (BMP) signalering. 27,28 Deze signaalroutes verhogen expressie van de transcriptiefactoren runt-gerelateerde transcriptiefactor 2 (Runx2) en osterix, die op hun beurt expressie van bot-gerelateerde eiwitten verhogen ( bijv., osteocalcine, sclerostine en alkalische fosfatase) in de vasculatuur die calcificatie bemiddelen 28-30 Wij en anderen hebben aangetoond dat de vasculaire calcificatie waargenomen in ApoE – / – en LDLR – / – muizen gevoed een vetrijk dieet en de spontane vasculaire calcificatie waargenomen in MGP – / – muizen zijn allemaal afhankelijk van het bot morfogenetische proteïne (BMP) signaling, en het is deze route die gericht hier. 11,25,31 BMPs potente osteogene factoren die nodig zijn voor botvorming en is bekend dat verhoogde expressie vertonen in menselijke atherosclerose. 32-34 In vitro studies hebben BMP signaaltransductie betrokken bij het ​​reguleren de expressie van osteogene factoren zoals Runx2. 35-37 Overexpressie van het ligand BMP, BMP-2, versnelt de ontwikkeling van vasculaire calcificatie in ApoE-deficiënte muizen die een vetrijke dieet. 38 Bovendien is het gebruik van specifieke remmers zoals BMP signalering als LDN-193189 (LDN) 39,40 en / of ALK3-Fc voorkomt de ontwikkeling van vasculaire calcificatie bij beide LDLR – / – muizen gevoed een vetrijk dieet en MGP-deficiënte muizen 11,25.

Vasculaire gladde spiercellen (VSMC's) spelen een cruciale rol in de ontwikkeling van vasculaire calcificatie. 30,41,42 De mediale vasculaire calcificatie die ontstaat in MGP-deficiënte muizen karakmerkt door een transdifferentiatie van VSMC een osteogene fenotype. Verlies van MGP resulteert in verminderde expressie van VSMC markers waaronder myocardin en alfa gladde spieren actine, met een daarmee gepaard gaande stijging van de osteogene merkers zoals Runx2 en osteopontine. Deze veranderingen samen met de ontwikkeling van vasculaire calcificatie. 25,43,44

Aorta calcificatie en ontsteking bij muizen worden kenmerkend bepaald met gebruikmaking histochemische technieken zoals alkalische fosfatase activiteit vroege calcificatie en osteogene activiteit, von Kossa en alizarine rood kleuring voor late calcificatie en immunohistochemische protocollen die macrofaag eiwit markers (bijv. Targeten, CD68, F4 / 80, Mac-1, MAC-2, MAC-3). 9,45 echter deze standaard beeldvormende technieken vereisen verwerking van de aorta weefsel in doorsneden, die tijdrovend en onvolmaakte door steekproefvertekening en zijn beperkt in hun vermogen om ontsteking en calcificat kwantificerenion in de gehele aorta. Dit protocol beschrijft een werkwijze voor het visualiseren en kwantificeren gehele aorta en middelgrote aderverkalking en macrofaag accumulatie gebruikmaking nabij infrarood fluorescentie (NIR) moleculaire beeldvorming ex vivo. Ook wordt een werkwijze voor het oogsten en het kweken van primaire aorta VSMC's van muizen en induceren van de verkalking van murine en menselijke VSMC's in vitro om de moleculaire mechanismen van vasculaire calcificatie bepalen. Deze technieken de onderzoeker zowel in vivo en in vitro methoden voor het bestuderen atherocalcific ziekte.

Protocol

Alle studies met muizen werden uitgevoerd in strikte overeenstemming met de aanbevelingen in de Gids voor de Zorg en gebruik van proefdieren van de National Institutes of Health. Huisvesting en alle procedures waarin muizen in deze studie beschreven, werden goedgekeurd door de Institutional Animal Care en gebruik Commissies van Massachusetts General Hospital (Subcommissie onderzoek Animal Care). Alle procedures werden uitgevoerd met zorg om het lijden te minimaliseren. 1. Bereiding van reagent…

Representative Results

Aorta verkalking in MGP – / – en wild-type muizen werd gemeten met behulp van beeldvorming van calcium NIR fluorescentie. Geen calcium NIR signaal werd gedetecteerd in de aorta's van wild-type muizen, waaruit blijkt dat zij calcificatie (figuur 2). Een sterk calcium NIR signaal werd gedetecteerd in de aorta van MGP-deficiënte muizen, wat overeenkomt met geavanceerde vasculaire calcificatie. Weefselcoupes van aorta's van wild-type en MGP – / -</su…

Discussion

Aderverkalking is een belangrijke risicofactor voor hart- en vaatziekten bij mensen en kunnen direct bijdragen aan de pathogenese van cardiovasculaire gebeurtenissen. 1,5,52 intima calciumafzetting in de dunne vezelige doppen van atherosclerose is voorgesteld om lokale biomechanische belasting bevorderen en zorgen plaquebreuk. 53,54 Mediale calcificatie effecten klinische resultaten door het verhogen van arteriële stijfheid, die cardiale hypertrofie kunnen veroorzaken en beïnvloeden de hartfuncti…

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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Tags

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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