JoVE Journal > Medicine
Summary
Abstract
Introduction
Protocol
Results
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Acknowledgements
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Automatic Translation
Medicine

Forkalkning af vaskulære glatte muskelceller og billeddannelse af aorta Forkalkning og Inflammation

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

Hjerte-kar-sygdom er den hyppigste årsag til sygelighed og dødelighed i verden, herunder USA, hvor den tegner sig for mere end 780,000 dødsfald årligt. 1 koronararterie forkalkning og aorta forkalkning er kendetegnende for aterosklerotisk sygdom og tjene som stærke prædiktorer for kardiovaskulære hændelser. 2- 4 To hovedtyper af vaskulær forkalkning er blevet rapporteret hos voksne: intima forkalkning, der er forbundet med åreforkalkning, og mediale (også kendt som Mönckeberg) forkalkning, der er forbundet med kronisk nyresygdom og diabetes fem af intima forkalkning sker i fastsættelsen af lipid ophobning og makrofag. infiltration i karvæggen. 5,6 Medial væg forkalkning sker uafhængigt af intimal forkalkning, lokaliseres til elastin fibre eller glatte muskelceller, og er ikke forbundet med lipidafsætning eller makrofag infiltration. 5,7,8 Undersøgelser på de molekylære mekanismer ivaskulær forkalkning har påberåbt sig cellebaserede og dyr modelsystemer. Gnavermodeller for atherocalcific sygdom omfatter mus deficiente i enten apolipoprotein E (ApoE) 9,10 eller low-density lipoprotein receptor (LDLR) 11 tilført en kost med højt fedtindhold, mens modeller for mediale forkalkning indbefatter mus med matrix Gla protein (MGP) mangel 12 eller rotter, der udvikler uræmi enten ved næsten total nefrektomi (5 / 6th nefrektomi modellen) eller ved udsættelse for en høj-adenin kost. 13

Her er modellen af ​​mediale vaskulær kalcifikation forbundet med MGP-mangel fokuseret på. MGP er et ekstracellulært protein, som inhiberer arteriel forkalkning. 12 mutationer i MGP genet er blevet identificeret i Keutel syndrom, en sjælden menneskelig sygdom karakteriseret ved diffus brusk forkalkning udover brachytelephalangy, høretab, og perifer pulmonal stenose. 14-18 Selvom ikke ofte observeret, 19koncentrisk forkalkning af flere arterier er blevet beskrevet i Keutel syndrom. 20 Fælles polymorfier i den humane MGP genet er forbundet med øget risiko for koronar arterie forkalkning, 21-23 mens højere cirkulerende niveauer af uncarboxylated, biologisk inaktivt MGP forudsige kardiovaskulær mortalitet. 24 I modsætning til mennesker med Keutel syndrom, MGP-deficiente mus udvikler en alvorlig vaskulær fænotype bestående af spontan udbredt arteriekalcifikation begyndende ved to ugers alderen og dø 6-8 uger efter fødslen på grund af aorta- brud. 12

I modsætning ApoE – / – og LDLR – / – mus fodret med en kost med højt fedtindhold, som udvikler intimale vaskulær kalcifikation med tilhørende makrofag-induceret inflammation, MGP – / -. Mus udvikler medial vaskulær kalcifikation i fravær af makrofaginfiltration 11,25 Selvom disse resultater tyder forskellige underliggende stimuli til Intimal og mediale forkalkning, der er overlapning i signaleringsmekanismer som medierer begge former for forkalkning. 26 Multiple signalveje er blevet identificeret, der bidrager til vaskulær kalcifikation herunder inflammatoriske mediatorer, såsom tumornekrosefaktor-α og IL-1 og pro-osteogene faktorer såsom Notch, Wnt, og knoglemorfogenetisk protein (BMP) signalering. 27,28 Disse signalveje forøge ekspression af transkriptionsfaktorer runt-relateret transkriptionsfaktor 2 (Runx2) og osterix, som igen øger ekspression af knoglerelaterede proteiner ( . f.eks osteocalcin, sclerostin, og alkalisk phosphatase) i vaskulaturen, der medierer forkalkning 28-30 Vi og andre har vist, at vaskulær kalcifikation observeret i ApoE – / – og LDLR – / – mus fodret med en kost med højt fedtindhold og den spontane vaskulær kalcifikation observeret i MGP – / – mus er alle afhængige af knoglemorfogenetisk protein (BMP) signaling, og det er denne vej, der er fokuseret på her. 11,25,31 BMP'er er potente osteogene faktorer, der kræves for knogledannelse og er kendt for at udvise forøget ekspression i human aterosklerose. 32-34 In vitro-undersøgelser har impliceret BMP-signalering i regulering ekspressionen af osteogene faktorer såsom Runx2. 35-37 Overekspression af BMP ligand, BMP-2, accelererer udviklingen af vaskulær kalcifikation i ApoE-deficiente mus fodret med en diæt med højt fedtindhold. 38 Endvidere er anvendelsen af specifikke BMP signalering inhibitorer, som LDN-193.189 (LDN) 39,40 og / eller alk3-Fc forhindrer udviklingen af vaskulær kalcifikation i både LDLR – / – mus fodret med en kost med højt fedtindhold og MGP-manglende mus 11,25.

Vaskulære glatte muskelceller (VSMC) har en afgørende rolle i udviklingen af vaskulær forkalkning. 30,41,42 Den mediale vaskulær forkalkning der udvikler sig i MGP-mangel mus er karakputerstyret ved en transdifferentiering af VSMC'er til et osteogent fænotype. Tab af MGP resulterer i nedsat ekspression af VSMC markører herunder myocardin og alfa glatmuskelactin, med en samtidig stigning i osteogene markører, såsom Runx2 og osteopontin. Disse ændringer falder sammen med udviklingen af vaskulær kalcifikation. 25,43,44

Aorta forkalkning og inflammation hos mus er typisk vurderet ved anvendelse histokemiske metoder, såsom alkalisk phosphatase aktivitet for tidlig forkalkning og osteogen aktivitet, von Kossa og alizarinrødt farvning for sent forkalkning, og immunohistokemiske protokoller, der er målrettet makrofag proteinmarkører (f.eks., CD68, F4 / 80, Mac-1, Mac-2, Mac-3). 9,45 Men disse standard billeddannende teknikker kræver behandling af aorta væv i tværsnit, hvilket er tidskrævende og ufuldkommen på grund af prøveudtagning bias, og er begrænset i deres evne til at kvantificere inflammation og calcification i hele aorta. Denne protokol beskriver en fremgangsmåde til at visualisere og kvantificere hele aorta og mellemstore arteriekalcifikation og makrofag akkumulering udnytte nær-infrarødt fluorescerende (NIR) molekylær billeddannelse ex vivo. Der tilvejebringes også en fremgangsmåde til høstning og dyrkning primære aortiske VSMC fra mus og inducere forkalkning af murine og humane VSMC in vitro for at bestemme de molekylære mekanismer, der ligger vaskulær kalcifikation. Disse teknikker giver investigator med både in vivo og in vitro metoder til at studere atherocalcific sygdom.

Protocol

Alle studier med mus blev udført i nøje overensstemmelse med anbefalingerne i Vejledning for Pleje og anvendelse af forsøgsdyr af National Institutes of Health. Boliger og alle procedurer, der involverer mus er beskrevet i denne undersøgelse blev godkendt af Institutional Animal Care og Brug udvalg af Massachusetts General Hospital (Underudvalget om Forskning Animal Care). Alle procedurer blev udført med omhu for at minimere lidelser. 1. Fremstilling af reagenser Near-Infrare…

Representative Results

Aorta forkalkning i MGP – / – og vildtype-mus blev målt ved anvendelse billeddannelse af calcium NIR fluorescens. Nr calcium NIR signal blev påvist i aorta fra vildtypemus, hvilket indikerer fravær af forkalkning (figur 2). En stærk calcium NIR signal blev påvist i aorta fra MGP-deficiente mus, hvilket er konsistent med avanceret vaskulær kalcifikation. Vævssnit af aorta fra vildtype og MGP – / – mus blev farvet med Alizarin red 25…

Discussion

Arteriel forkalkning er en vigtig risikofaktor for hjerte-kar-sygdom hos mennesker og kan bidrage direkte til patogenesen af kardiovaskulære hændelser. 1,5,52 intima calcium deposition i de tynde fibrøse hætter af aterosklerotisk sygdom er blevet foreslået at øge den lokale biomekanisk stress og bidrage til plaque ruptur. 53,54 Medial forkalkning påvirkninger kliniske resultater ved at øge arteriel stivhed, som kan fremkalde hjertehypertrofi og påvirke hjertefunktionen. 55 Derfo…

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

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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