The Extracellular Matrix: Methods and Protocols

282 Pages·2019·7.43 MB·English

by  Davide Vigetti, Achilleas D. Theocharis

Methods in
Molecular Biology 1952
Davide Vigetti
Achilleas D. Theocharis Editors
The
Extracellular
Matrix
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire,AL109AB,UK
Forfurther volumes:
http://www.springer.com/series/7651
The Extracellular Matrix
Methods and Protocols
Edited by
Davide Vigetti
Department of Medicine and Surgery, University of Insubria, Varese, Italy
Achilleas D. Theocharis
Department of Chemistry, University of Patras, Patras, Greece
Editors
DavideVigetti AchilleasD.Theocharis
DepartmentofMedicineandSurgery DepartmentofChemistry
UniversityofInsubria UniversityofPatras
Varese,Italy Patras,Greece
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-4939-9132-7 ISBN978-1-4939-9133-4 (eBook)
https://doi.org/10.1007/978-1-4939-9133-4
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Preface
Extracellular matrix (ECM) is a three-dimensional noncellular network where cells reside.
Apart from structural support, ECM controls cell phenotype and functions in tissues and
organs.ECMcomponentsarecomposedoffibrillarproteinsincludingcollagens,fibronec-
tin, laminins and elastin, proteoglycans, hyaluronan, and several glycoproteins including
matricellular proteins. All these ECM molecules interact with each other as well as with
certaincellsurfacereceptorscreatingcomplexbutwell-organizednetworks.Specifically,the
elements of the ECMs can influence tissue homeostasis, cell proliferation, migration, sur-
vival, differentiation, angiogenesis, immune function, and autophagy. ECM is subjected in
continuous remodeling in physiological and pathologic circumstances. ECM-degrading
enzymes including matrix metalloproteinases and hyaluronidases are responsible for ECM
remodeling, whereas resident cells synthesize and deposit newly formed ECM material to
reconstitutethephysiologictissuearchitecture.ECMremodelingisregulatedbycell-matrix
and cell-cell communication which are mediated by direct and indirect interactions.
Recently, exosomes have been demonstrated to be integral components of ECM that are
involved in matrix remodeling and cell-matrix and cell-cell communication. Abnormal
remodeling of ECM often occurs and is associated with disease development and progres-
sion. Researchers have focus on ECM molecules to develop new therapeutic modalities,
drug delivery approaches, and biomaterials. Nowadays, ECM is considered as a regulatory
element of tissues and organs and not only as a niche for cells. It controls cell fate and
functions, and the research on matrix biology becomes equally important with that of cell
biology.
The aim of this volume of the Methods in Molecular Biology series is to provide readers
withestablishedexperimentalprotocolsusefulfortheisolation,characterization,anddetec-
tion of ECM molecules as well as methods to study the activity and role of ECM compo-
nentsonvariousbiologicalfunctions,theformationofexosomesandtissuesurrogates.This
volume is aimed at basic and clinical researchers including biochemists, cell and molecular
biologists,pathologists,oncologists,andphysicians.Thedetailedprotocolsandapplications
presentedwithinthisbookseriescanbeexploitedbyPh.D.studentsandseniorresearchers
tocarryoutresearchinthefieldofextracellularmatrixbiology.Thisbookcontains21chap-
ters that display a variety of protocols ranging from biochemical and cell and molecular
biologyassaystocomplextissueimagingtechniquesandinvivomodelstoelucidatetherole
of extracellular matrix. Chapter 1 provides a short introduction in ECM structure and its
regulatory role on cell functions and behavior. It emphasizes on the complex interplay
between ECM components and cells that controls tissue homeostasis and progression of
diseases. The gene expression of ECM molecules directly on fixed tissues is described in
Chap. 2. This easy to perform and convenient method allows the recognition of cells
expressinga certaincomponent and canbejoined withimmunostainingin thesame tissue
samples to demonstrate the biosynthesis of ECM in situ. Protocols on modern methods
including transmission electron microscope, scanning electron microscope, and atomic
forcemicroscopetovisualizethesupramolecularassemblyofcollagenintissuesareprovided
in Chap. 3. Chapter 4 provides protocols for studying the nature, the posttranslational
modifications, and the kinetic of secretion of collagen in vitro by using steady-state and
pulse-chase labeling with 3H-proline. Protocols for the isolation and characterization of
v
vi Preface
marine invertebrate-derived glycosaminoglycans as well as for the analysis of proteoglycan
biosynthesis and secretion in vitro are presented in Chaps. 5 and 6. In Chap. 7, the
methodology for the analysis of precursor UDP-sugars for the biosynthesis of various
cellular glycans and glycoconjugates is described. It involves isolation from cultured cells
and tissues utilizing ion-pair solid-phase extraction with graphitized carbon cartridges and
subsequentanalysisusinganion-exchangehigh-performanceliquidchromatography.Chap-
ters8and9presentmethodsfor thedeterminationofhyaluronanmolecularmassutilizing
simpleelectrophoresisandvisualizationofpericellularhyaluronanbyaquicksemiquantita-
tiveparticleexclusionassayandvalidationbyfluorescencedetection.Themethodologyfor
the determination of cell surface-associated hyaluronan by flow cytometry using
hyaluronan-binding proteins conjugated to different fluorochromes is described in
Chap.10.InChap.11,aprotocolforisolationofprimaryculturesofadultratsmallintestine
myenteric ganglia and subsequent visualization of hyaluronan is provided. A simple and
sensitive approach for analysis of hyaluronidases present in biological fluids employing
hyaluronan zymography and microtiter plate assay is given in Chap. 12. Chapter 13
describes a protocol for studying the effect of glycosaminoglycans on voltage-dependent
ion channel gating. Comprehensive methodology for monitoring matrix-induced autop-
hagy utilizing morphological and microscopic characterization, biochemical analysis, and
signalingpathwayinvestigationaswellasfordetectingmitophagyviamonitoringchangesin
mitochondrial DNA and permeability is provided in Chap. 14. In Chaps. 15 and 16,
experimental approaches for determining gelatinolytic activity in tissues by in situ zymo-
graphyandreal-timegelatinzymographyaredescribed.Chapters17and18presentmeth-
odologies for evaluation of angiogenesis by using in vitro and in vivo models. Chapter 19
includesaprotocolforisolationandcharacterizationofexosomesreleasedinthecellculture
medium.Themethodologyfor thepreparationandcharacterizationofrichinextracellular
matrixtissuesurrogatesfromvariouscellpopulationsusingtheprinciplesofmacromolecu-
lar crowding is described in Chap. 20. In addition, Chap. 21 discusses strategies to target
ECM components for disease treatment. It proposes the creation of a pioneer ECM
multidisciplinary integrated platform in order to decipher ECM homeostasis that can lead
tothedevelopmentoftherapeuticnewagents,protein-basedinhibitors,diagnostics,medi-
caldevices,artificialtissues,anddrugdeliverystrategies.
We hope that this volume will beuseful and enjoyable for readers and it will foster the
advancement of research on extracellular matrix biology. We are very appreciative of all
contributionsprovidedbyeminentscientistsinthefield.WearealsogratefultoProf.John
M. Walker, editor-in-chief of Methods in Molecular Biology series, for the continuous
interest and support throughout this period that helped us to accomplish the editing of
thisvolume.
Varese,Italy DavideVigetti
Patras,Greece AchilleasD.Theocharis
Contents
Preface ..................................................................... v
Contributors................................................................. ix
1 TheComplexInterplayBetweenExtracellularMatrixandCellsinTissues ..... 1
DimitraManou,IlariaCaon,PanagiotisBouris,
Irene-EvaTriantaphyllidou,CristinaGiaroni,AlbertoPassi,
NikosK.Karamanos,DavideVigetti,andAchilleasD.Theocharis
2 MethodforStudyingECMExpression:InSituRT-PCR .................... 21
ElenaCarav`a,CristianaMarcozzi,BarbaraBartolini,
MarcellaReguzzoni,PaolaMoretto,IlariaCaon,
EvgeniaKarousou,AlbertoPassi,andManuelaViola
3 VisualizingtheSupramolecularAssemblyofCollagen ....................... 33
MarioRaspanti,MarcellaReguzzoni,PetraRitaBasso,
MarinaProtasoni,andDe´sire´eMartini
4 Steady-StateandPulse-ChaseAnalysesofFibrillarCollagen.................. 45
AntonellaForlino,FrancescaTonelli,andRobertaBesio
5 MethodsforIsolationandCharacterizationofSulfated
GlycosaminoglycansfromMarineInvertebrates............................. 55
MarianaP.Stelling,AnandaA.deBento,PhilippeCaloba,
EduardoVilanova,andMauroS.G.Pava˜o
6 AnalysisofProteoglycanSynthesisandSecretioninCellCultureSystems...... 71
ChiaraPaganini,RossellaCostantini,andAntonioRossi
7 AnalysisofUDP-SugarsfromCulturedCellsandSmallTissueSamples........ 81
SannaOikariandMarkkuI.Tammi
8 MethodsforHyaluronanMolecularMassDeterminationbyAgarose
GelElectrophoresis ..................................................... 91
MaryK.Cowman
9 HyaluronanPericellularMatrix:ParticleExclusionAssay..................... 103
MelanieA.Simpson
10 DeterminationofCell-SurfaceHyaluronanThroughFlowCytometry......... 111
DaianaL.Vitale,FiorellaM.Spinelli,andLauraAlaniz
11 MethodforDetectingHyaluronaninIsolatedMyentericPlexusGanglia
ofAdultRatSmallIntestine.............................................. 117
MichelaBistoletti,PaolaMoretto,andCristinaGiaroni
12 AnalyzingHyaluronidasesinBiologicalFluids.............................. 127
ChristosVelesiotis,StellaVasileiou,andDemitriosH.Vynios
13 MethodforStudyingthePhysicalEffectofExtracellularMatrix
onVoltage-DependentIonChannelGating................................ 143
EleonoraSolariandAndreaMoriondo
14 MethodsforMonitoringMatrix-InducedAutophagy ....................... 157
CarolynChen,AasthaKapoor,andRenatoV.Iozzo
vii
viii Contents
15 MethodforDeterminingGelatinolyticActivityinTissue:InSitu
GelatinZymography .................................................... 193
ElinHadler-OlsenandJan-OlofWinberg
16 MethodforDeterminingGelatinolyticActivityinTissueExtracts:
Real-TimeGelatinZymography .......................................... 201
ElinHadler-OlsenandJan-OlofWinberg
17 InVitroSpheroidSproutingAssayofAngiogenesis ......................... 211
FatemaTuzZahra,EfrossiniCholeva,MdSanaullahSajib,
EvangeliaPapadimitriou,andConstantinosM.Mikelis
18 MatrigelPlugAssayforInVivoEvaluationofAngiogenesis.................. 219
PinelopiKastana,FatemaTuzZahra,DespoinaNtenekou,
StamatikiKatraki-Pavlou,DimitrisBeis,MichailS.Lionakis,
ConstantinosM.Mikelis,andEvangeliaPapadimitriou
19 ExosomesfromCellCulture-ConditionedMedium:Isolation
byUltracentrifugationandCharacterization................................ 233
AnuragPurushothaman
20 PreparationandCharacterizationofTissueSurrogatesRich
inExtracellularMatrixUsingthePrinciplesofMacromolecular
Crowding.............................................................. 245
AdrianDjalali-Cuevas,SergioGarnica-Galvez,AndreaRampin,
DianaGaspar,IoannisSkoufos,AthinaTzora,NikitasPrassinos,
NikolaosDiakakis,andDimitriosI.Zeugolis
21 NovelApproachesforExtracellularMatrixTargetinginDisease
Treatment ............................................................. 261
NikolaosA.AfratisandIritSagi
Index ...................................................................... 277
Contributors
NIKOLAOSA.AFRATIS (cid:1) DepartmentofBiologicalRegulation,WeizmannInstituteofScience,
Rehovot,Israel
LAURAALANIZ (cid:1) LaboratoriodeMicroambienteTumoral,UniversidadNacionaldelNoroeste
delaPcia.deBs.As,CentrodeInvestigacionesyTransferenciadelNoroestedelaProvincia
deBuenosAires(CITNOBAUNNOBA-CONICET),Junı´n,BuenosAires,Argentina
BARBARABARTOLINI (cid:1) DepartmentofMedicineandSurgery,UniversityofInsubria,Varese,
Italy
PETRA RITABASSO (cid:1) DepartmentofMedicineandSurgery,UniversityofInsubria,Varese,
Italy
DIMITRISBEIS (cid:1) BiomedicalResearchFoundationAcademyofAthens,Athens,Greece
ROBERTABESIO (cid:1) BiochemistryUnit,DepartmentofMolecularMedicine,Universityof
Pavia,Pavia,Italy
MICHELABISTOLETTI (cid:1) DepartmentofMedicineandSurgery,UniversityofInsubria,Varese,
Italy
PANAGIOTISBOURIS (cid:1) LaboratoryofBiochemistry,DepartmentofChemistry,Universityof
Patras,Patras,Greece
PHILIPPECALOBA (cid:1) Laborat(cid:1)oriodeBioquı´micaeBiologiaCelulardeGlicoconjugados,
ProgramadeGlicobiologia,InstitutodeBioquı´micaMe´dicaLeopoldodeMeisandHospital
Universita´rioClementinoFragaFilho,UniversidadeFederaldoRiodeJaneiro,Riode
Janeiro,Brazil;InstitutoFederaldeEducac¸a˜o,CieˆnciaeTecnologiadoRiodeJaneiro,Rio
deJaneiro,Brazil
ILARIACAON (cid:1) DepartmentofMedicineandSurgery,UniversityofInsubria,Varese,Italy
ELENACARAVA` (cid:1) DepartmentofMedicineandSurgery,UniversityofInsubria,Varese,Italy
CAROLYNCHEN (cid:1) DepartmentofPathology,AnatomyandCellBiologyandtheCancerCell
BiologyandSignalingProgram,SidneyKimmelMedicalCollegeatThomasJefferson
University,Philadelphia,PA,USA
EFROSSINICHOLEVA (cid:1) LaboratoryofMolecularPharmacology,DepartmentofPharmacy,
SchoolofHealthSciences,UniversityofPatras,Patras,Greece
ROSSELLACOSTANTINI (cid:1) DepartmentofMolecularMedicine,BiochemistryUnit,Universityof
Pavia,Pavia,Italy
MARYK.COWMAN (cid:1) NewYorkUniversity,TandonSchoolofEngineering,NewYork,NY,
USA
ANANDA A.DEBENTO (cid:1) Laborat(cid:1)oriodeBioquı´micaeBiologiaCelulardeGlicoconjugados,
ProgramadeGlicobiologia,InstitutodeBioquı´micaMe´dicaLeopoldodeMeisandHospital
Universita´rioClementinoFragaFilho,UniversidadeFederaldoRiodeJaneiro,Riode
Janeiro,Brazil;InstitutoFederaldeEducac¸a˜o,CieˆnciaeTecnologiadoRiodeJaneiro,Rio
deJaneiro,Brazil
NIKOLAOSDIAKAKIS (cid:1) SchoolofVeterinaryMedicine,AristotleUniversityofThessaloniki,
Thessaloniki,Greece
ADRIAN DJALALI-CUEVAS (cid:1) BioprocessingandBiotechnologyLaboratory,Departmentof
AgriculturalTechnology,TechnologicalEducationInstitute(TEI)ofEpirus,Arta,Greece;
SchoolofVeterinaryMedicine,AristotleUniversityofThessaloniki,Thessaloniki,Greece;
ix
x Contributors
Regenerative,ModularandDevelopmentalEngineeringLaboratory(REMODEL),
NationalUniversityofIrelandGalway(NUIGalway),Galway,Ireland
ANTONELLAFORLINO (cid:1) BiochemistryUnit,DepartmentofMolecularMedicine,Universityof
Pavia,Pavia,Italy
SERGIOGARNICA-GALVEZ (cid:1) BioprocessingandBiotechnologyLaboratory,Departmentof
AgriculturalTechnology,TechnologicalEducationInstitute(TEI)ofEpirus,Arta,Greece;
SchoolofVeterinaryMedicine,AristotleUniversityofThessaloniki,Thessaloniki,Greece;
Regenerative,ModularandDevelopmentalEngineeringLaboratory(REMODEL),
NationalUniversityofIrelandGalway(NUIGalway),Galway,Ireland
DIANA GASPAR (cid:1) Regenerative,ModularandDevelopmentalEngineeringLaboratory
(REMODEL),NationalUniversityofIrelandGalway(NUIGalway),Galway,Ireland;
ScienceFoundationIreland(SFI)CentreforResearchinMedicalDevices(CU´RAM),
NationalUniversityofIrelandGalway(NUIGalway),Galway,Ireland
CRISTINAGIARONI (cid:1) DepartmentofMedicineandSurgery,UniversityofInsubria,Varese,
Italy
ELINHADLER-OLSEN (cid:1) FacultyofHealthSciences,DepartmentofMedicalBiology,UiT—The
ArcticUniversityofNorway,Tromsø,Norway;FacultyofHealthSciences,Departmentof
ClinicalDentistry,UiT—TheArcticUniversityofNorway,Tromsø,Norway;Department
ofClinicalPathology,UniversityHospitalofNorthNorway,Tromsø,Norway
RENATOV.IOZZO (cid:1) DepartmentofPathology,AnatomyandCellBiologyandtheCancerCell
BiologyandSignalingProgram,SidneyKimmelMedicalCollegeatThomasJefferson
University,Philadelphia,PA,USA
AASTHAKAPOOR (cid:1) DepartmentofPathology,AnatomyandCellBiologyandtheCancerCell
BiologyandSignalingProgram,SidneyKimmelMedicalCollegeatThomasJefferson
University,Philadelphia,PA,USA
NIKOSK.KARAMANOS (cid:1) LaboratoryofBiochemistry,DepartmentofChemistry,Universityof
Patras,Patras,Greece
EVGENIAKAROUSOU (cid:1) DepartmentofMedicineandSurgery,UniversityofInsubria,Varese,
Italy
PINELOPIKASTANA (cid:1) LaboratoryofMolecularPharmacology,DepartmentofPharmacy,School
ofHealthSciences,UniversityofPatras,Patras,Greece
STAMATIKIKATRAKI-PAVLOU (cid:1) LaboratoryofMolecularPharmacology,Departmentof
Pharmacy,SchoolofHealthSciences,UniversityofPatras,Patras,Greece;Biomedical
ResearchFoundationAcademyofAthens,Athens,Greece
MICHAILS.LIONAKIS (cid:1) FungalPathogenesisSection,LaboratoryofClinicalImmunologyand
Microbiology(LCIM),NationalInstituteofAllergy&InfectiousDiseases(NIAID),
NationalInstitutesofHealth(NIH),Bethesda,MD,USA
DIMITRA MANOU (cid:1) LaboratoryofBiochemistry,DepartmentofChemistry,Universityof
Patras,Patras,Greece
CRISTIANAMARCOZZI (cid:1) DepartmentofMedicineandSurgery,UniversityofInsubria,Varese,
Italy
DE´SIRE´EMARTINI (cid:1) DepartmentofBiomedicalandNeuromotorSciences,Universityof
Bologna,Bologna,Italy
CONSTANTINOSM.MIKELIS (cid:1) DepartmentofPharmaceuticalSciences,SchoolofPharmacy,
TexasTechUniversityHealthSciencesCenter,Amarillo,TX,USA
PAOLAMORETTO (cid:1) DepartmentofMedicineandSurgery,UniversityofInsubria,Varese,Italy
ANDREA MORIONDO (cid:1) DepartmentofMedicineandSurgery,UniversityofInsubria,Varese,
Italy